Methods for treating disease and forming a supplemented fibrin matrix

ABSTRACT

This invention provides supplemented and unsupplemented tissue sealants as well as methods for their production and use thereof. Disclosed are tissue sealants supplemented with at least one oligonucleotide. The composition may be further supplemented with, for example, one or more analgesics, antimicrobial compositions, anticoagulants, antiproliferatives, anti-inflammatory compounds, cytokines, cytotoxins, drugs, growth factors, interferons, hormones, lipids, demineralized bone or bone morphogenetic proteins, cartilage inducing factors, oligonucleotides polymers, polysaccharides, polypeptides, protease inhibitors, vasoconstrictors or vasodilators, vitamins, minerals, stabilizers and the like.

This application is a Continuation-in-Part application of U.S.application Ser. No. 08/351,006, filed Dec. 7, 1994, now abandoned,which is a Continuation-in-Part application of U.S. application Ser. No.08/328,552, filed Oct. 25, 1994, now abandoned, which is a Continuationapplication of U.S. application Ser. No. 08/031,164, filed Mar. 12,1993, abandoned, which is a Continuation-in-Part application of U.S.application Ser. Nos. 07/618,419 and 07/798,919, filed Nov. 27, 1990,and Nov. 27, 1991, respectively, both of which are abandoned, all ofwhich are herein incorporated by reference.

RIGHTS OF THE UNITED STATES GOVERNMENT IN THIS INVENTION

Under a Cooperative Research and Development Agreement between TheAmerican National Red Cross and The U.S. Army Institute of DentalResearch, the U.S. Government may have a non-exclusive, irrevocable,paid-up license in one or more embodiments of this invention.

FIELD OF INVENTION

This invention is directed to unsupplemented and supplemented TissueSealants (TS), such as fibrin glue (FG), as well as to methods of theirproduction and use. In one embodiment, this invention is directed to TSswhich do not inhibit full-thickness skin wound healing. In anotherembodiment, this invention is directed to TSs which have beensupplemented with a growth factor(s) and/or a drug(s), as well as tomethods of their production and use. The particular growth factor(s) ordrug(s) that is selected is a function of its use.

BACKGROUND ART

A. Wound Healing and Growth Factors

Wound healing, the repair of lesions, begins almost instantly afterinjury. It requires the successive coordinated function of a variety ofcells and the close regulation of degradative and regenerative steps.The proliferation, differentiation and migration of cells are importantbiological processes which underlie wound healing, which also involvesfibrin clot formation, resorption of the clot, tissue remodeling, suchas fibrosis, endothelialization and epithelialization. Wound healinginvolves the formation of highly vascularized tissue that containsnumerous capillaries, many active fibroblasts, and abundant collagenfibrils, but not the formation of specialized skin structures.

The process of wound healing can be initiated by thromboplastin whichflows out of injured cells. Thromboplastin contacts plasma factor VII toform factor X activator, which then, with factor V and in a complex withphospholipids and calcium, converts prothrombin into thrombin. Thrombincatalyzes the release of fibrinopeptides A and B from fibrinogen toproduce fibrin monomers, which aggregate to form fibrin filaments.Thrombin also activates the transglutaminase, factor XIIIa, whichcatalyzes the formation of isopeptide bonds to covalently cross-link thefibrin filaments. Alpha₂-antiplasmin is then bound by factor XIII ontothe fibrin filaments to thereby protect the filaments from degradationby plasmin (see, for example, Doolittle et al., Ann. Rev. Biochem.53:195–229 (1984)).

When a tissue is injured, polypeptide growth factors, which exhibit anarray of biological activities, are released into the wound where theyplay a crucial role in healing (see, e.g., Hormonal Proteins andPeptides (Li, C. H., ed.) Volume 7, Academic Press, Inc., New York, N.Y.pp. 231–277 (1979) and Brunt et al., Biotechnology 6:25–30 (1988)).These activities include recruiting cells, such as leukocytes andfibroblasts, into the injured area, and inducing cell proliferation anddifferentiation. Growth factors that may participate in wound healinginclude, but are not limited to: platelet-derived growth factors(PDGFs); insulin-binding growth factor-1 (IGF-1); insulin-binding growthfactor-2 (IGF-2); epidermal growth factor (EGF); transforming growthfactor-α (TGF-α); transforming growth factor-β (TGF-β); platelet factor4 (PF-4); and heparin binding growth factors one and two (HBGF-1 andHBGF-2, respectively).

PDGFs are stored in the alpha granules of circulating platelets and arereleased at wound sites during blood clotting (see, e.g., Lynch et al.,J. Clin. Invest. 84:640–646 (1989)). PDGFs include: PDGF; plateletderived angiogenesis factor (PDAF); TGF-β; and PF-4, which is achemoattractant for neutrophils (Knighton et al., in Growth Factors andOther Aspects of Wound Healing: Biological and Clinical Implications,Alan R. Liss, Inc., New York, N.Y., pp. 319–329 (1988)). PDGF is amitogen, chemoattractant and a stimulator of protein synthesis in cellsof mesenchymal origin, including fibroblasts and smooth muscle cells.PDGF is also a nonmitogenic chemoattractant for endothelial cells (see,for example, Adelmann-Grill et al., Eur. J. Cell Biol. 51:322–326(1990)).

IGF-1 acts in combination with PDGF to promote mitogenesis and proteinsynthesis in mesenchymal cells in culture. Application of either PDGF orIGF-1 alone to skin wounds does not enhance healing, but application ofboth factors together appears to promote connective tissue andepithelial tissue growth (Lynch et al., Proc. Natl. Acad. Sci.76:1279–1283 (1987)).

TGF-β is a chemoattractant for macrophages and monocytes. Depending uponthe presence or absence of other growth factors, TGF-β may stimulate orinhibit the growth of many cell types. For example, when applied invivo, TGF-β increases the tensile strength of healing dermal wounds.TGF-β also inhibits endothelial cell mitosis, and stimulates collagenand glycosaminoglycan synthesis by fibroblasts.

Other growth factors, such as EGF, TGF-α, the HBGFs and osteogenin arealso important in wound healing. EGF, which is found in gastricsecretions and saliva, and TGF-α, which is made by both normal andtransformed cells, are structurally related and may recognize the samereceptors. These receptors mediate proliferation of epithelial cells.Both factors accelerate reepithelialization of skin wounds. ExogenousEGF promotes wound healing by stimulating the proliferation ofkeratinocytes and dermal fibroblasts (Nanney et al., J. Invest.Dermatol. 83:385–393 (1984) and Coffey et al., Nature 328:817–820(1987)). Topical application of EGF accelerates the rate of healing ofpartial thickness wounds in humans (Schultz et al., Science 235:350–352(1987)). Osteogenin, which has been purified from demineralized bone,appears to promote bone growth (see, e.g., Luyten et al., J. Biol. Chem.264:13377 (1989)). In addition, platelet-derived wound healing formula,a platelet extract which is in the form of a salve or ointment fortopical application, has been described (see, e.g., Knighton et al.,Ann. Surg. 204:322–330 (1986)).

The Heparin Binding Growth Factors (HBGFs), also known as FibroblastGrowth Factors (FGFs), which include acidic HBGF (aHBGF also known asHBFG-1 or FGF-1) and basic HBGF (bHBGF also known as HBGF-2 or FGF-2),are potent mitogens for cells of mesodermal and neuroectodermallineages, including endothelial cells (see, e.g., Burgess et al., Ann.Rev. Biochem. 58:575–606 (1989)). In addition, HBGF-1 is chemotactic forendothelial cells and astroglial cells. Both HBGF-1 and HBGF-2 bind toheparin, which protects them from proteolytic degradation. The array ofbiological activities exhibited by the HBGFs suggests that they play animportant role in wound healing.

Basic fibroblast growth factor (FGF-2) is a potent stimulator ofangiogenesis and the migration and proliferation of fibroblasts (see,for example, Gospodarowicz et al., Mol. Cell. Endocinol. 46:187–204(1986) and Gospodarowicz et al., Endo. Rev. 8:95–114 (1985)). Acidicfibroblast growth factor (FGF-1) has been shown to be a potentangiogenic factor for endothelial cells (Burgess et al., supra, 1989).However, it has not been established if any FGF growth factor ischemotactic for fibroblasts.

Growth factors are, therefore, potentially useful for specificallypromoting wound healing and tissue repair. However, their use to promotewound healing has yielded inconsistent results (see, e.g., Carter etal., in Growth Factors and Other Aspects of Wound Healing: Biologicaland Clinical Implications, Alan R. Liss, Inc., New York, N.Y., pp.303–317 (1988)). For example, PDGF, IGF-1, EGF, TGF-α, TGF-β and FGF(also known as HBGF) applied separately to standardized skin wounds inswine had little effect on the regeneration of connective tissue orepithelium in the wounds (Lynch et al., J. Clin. Invest. 84:640–646(1989)). Of the factors tested, TGF-β stimulated the greatest responsealone. However, a combination of factors, such as PDGF-bb homodimer andIGF-1 or TGF-α produced a dramatic increase in connective tissueregeneration and epithelialization. (Id.) Tsuboi et al. have reportedthat the daily application of bFGF to an open wound stimulated woundhealing in healing-impaired mice but not in normal mice (J. Exp. Med.172:245–251 (1990)). On the other hand, the application to human skinwounds of crude preparations of porcine or bovine platelet lysate, whichpresumably contained growth factors, increased the rate at which thewounds closed, the number of cells in the healing area, the growth ofblood vessels, the total rate of collagen deposition and the strength ofthe scar tissue (Carter et al., supra).

The reasons for such inconsistent results are not known, but might bethe result of difficulty in applying growth factors to a wound in amanner in which they can exhibit their normal array of biologicalactivities. For example, it appears that some growth factor receptorsmust be occupied for at least 12 hours to produce a maximal biologiceffect (Presta et al., Cell Regul. 2:719–726 (1991) and Rusnati et al.,J. Cell Physiol. 154:152–161 (1993)). Because of such inconsistentresults, the role played by exogenously applied growth factors instimulating wound healing is not clear. Further, a means by which growthfactors might be applied to wounds to produce prolonged contact betweenthe wound and the growth factor(s) is not presently known.

B. TSs

Surgical adhesives and TSs which contain plasma proteins are used forsealing internal and external wounds, such as in bones and skin, toreduce blood loss and maintain hemostasis. Such TSs contain bloodclotting factors and other blood proteins. FG, also called fibrinsealant, is a gel similar to a natural clot which is prepared fromplasma. The precise components of each FG are a function of theparticular plasma fraction which is used as a starting material.Fractionation of plasma components can be effected by standard proteinpurification methods, such as ethanol, polyethylene glycol, and ammoniumsulfate precipitation, ion exchange, and gel filtration chromatography.Typically FG contains a mixture of proteins including traces of albumin,fibronectin and plasminogen. In Canada, Europe and possibly elsewhere,commercially available FG typically also contains aprotinin as astabilizer.

FGs generally are prepared from: (1) a fibrinogen concentrate, whichcontains fibronectin, Factor XIII, and von Willebrand factor; (2) driedhuman or bovine thrombin; and (3) calcium ions. Commercially preparedFGs generally contain bovine components. The fibrinogen concentrate canbe prepared from plasma by cryoprecipitation followed by fractionation,to yield a composition that forms a sealant or clot upon mixture withthrombin and an activator of thrombin such as calcium ions. Thefibrinogen and thrombin concentrates are prepared in lyophilized formand are mixed with a solution of calcium chloride immediately prior touse. Upon mixing, the components are applied to a tissue where theycoagulate on the tissue surface and form a clot that includescross-linked fibrin. Factor XIII, which is present in the fibrinogenconcentrate, catalyzes the cross-linking.

Australian Patent 75097/87 describes a one-component adhesive, whichcontains an aqueous solution of fibrinogen, factor XIII, a thrombininhibitor, such as antithrombin III, prothrombin factors, calcium ions,and, if necessary, a plasmin inhibitor. Stroetmann, U.S. Pat. Nos.4,427,650 and 4,427,651, describes the preparation of an enriched plasmaderivative in the form of a powder or sprayable preparation for enhancedwound closure and healing that contains fibrinogen, thrombin and/orprothrombin, and a fibrinolysis inhibitor, and may also contain otheringredients, such as a platelet extract. Rose et al., U.S. Pat. Nos.4,627,879 and 4,928,603, disclose methods for preparing cryoprecipitatedsuspensions that contain fibrinogen and Factor XIII and their use toprepare a FG. JP 1-99565 discloses a kit for the preparation of fibrinadhesives for wound healing. Alterbaum (U.S. Pat. No. 4,714,457) andMorse et al. (U.S. Pat. No. 5,030,215) disclose methods to produceautologous FG. In addition, improved FG delivery systems have beendisclosed elsewhere (Miller et al., U.S. Pat. No. 4,932,942 and Morse etal., PCT Application WO 91/09641).

IMMUNO AG (Vienna, Austria) and BEHRINGWERKE AG (Germany) (Gibble etal., Transfusion 30:741–747 (1990)) presently have FGs on the market inEurope and elsewhere (see, e.g., U.S. Pat. Nos. 4,377,572 and 4,298,598,which are owned by IMMUNO AG). TSs are not commercially available in theU.S. However, the American National Red Cross and BAXTER/HYLAND (LosAngeles, Calif.) have recently co-developed a FG (ARC/BH FG) which isnow in clinical studies.

The TSs which are used clinically outside of the U.S. pose certainclinical risks and have not been approved by the Food and DrugAdministration for use in the USA. For example, the TSs available inEurope contain proteins of non-human origin such as aprotinin and bovinethrombin. Since these proteins are of non-human origin, people maydevelop allergic reactions to them. In Europe heat inactivation is usedto inactivate viruses which may be present in the components of the FG.However, this heat inactivation method may produce denatured proteins inthe FG which may also be allergenic. In addition, there is concern thatthis inactivation method will not inactivate prions which cause bovinespongiform encephalopathy, “mad cow disease,” which may be present inthe TS due to the use of bovine proteins therein. Since this diseaseappears to have already crossed from sheep, in which it is called“scrapies,” to cows, it is not an insignificant concern that it couldinfect humans.

The ARC/BH FG has advantages over the TSs available in Europe because itdoes not contain bovine proteins. For example, the ARC/BH TS containshuman thrombin instead of bovine thrombin and does not containaprotinin. Since the ARC/BH FG does not contain bovine proteins itshould be less allergenic in humans than those TSs available in Europe.In addition, the ARC/BH FG is virally inactivated by a solvent detergentmethod which produces fewer denatured proteins and thus is lessallergenic than those available in Europe. Therefore, the ARC/BH FGpossesses advantages over the TSs which are now commercially availablein other countries.

FG is primarily formulated for clinical topical application and is usedto control bleeding, maintain hemostasis and promote wound healing. Theclinical uses of FG have recently been reviewed (Gibble et al.,Transfusion 30:741–747 (1990); Lerner et al., J. Surg. Res. 48:165–181(1990)). By sealing tissues FG prevents air or fluid leaks, induceshemostasis, and may contribute to wound healing indirectly by reducingor preventing events which may interfere with wound healing such asbleeding, hematomas, infections, etc. Although FG maintains hemostasisand reduces blood loss, it has not yet been shown to possess true woundhealing properties. Because FG is suitable for both internal andexternal injuries, such as bone and skin injuries, and is useful tomaintain hemostasis, it is desirable to enhance its wound healingproperties.

FG with a fibrinogen concentration of approximately 39 g/l and athrombin concentration of 200–600 U/ml has produced clots withsignificantly increased stress, energy absorption and elasticity values(Byrne et al., Br. J. Surg. 78:841–843 (1991)). Perforated Tefloncylinders filled with fibrin clot (5 mg/ml) and implanted subcutaneouslystimulated the formation of granulation tissue, including an increasedprecipitation of collagen, when compared to empty cylinders (Hedelin etal., Eur. Surg. Res. 15:312 (1983)).

C. Bone Wounds and Their Repair

The sequence of bone induction was first described by Urist et al. usingdemineralized cortical bone matrix (Clin. Orthop. Rel. Res. 71:271(1970) and Proc. Natl. Acad. Sci. USA 70:3511 (1973)). Implantedsubcutaneously in allogeneic recipients, demineralized cortical bonematrix releases factors which act as local mitogens to stimulate theproliferation of mesenchymal cells (Rath et al., Nature (Lond.) 278:855(1979)). New bone formation occurs between 12 and 18 dayspostimplantation. Ossicle development replete with hematopoietic marrowlineage occurred by day 21 (Reddi, A., In Extracellular MatrixBiochemistry (Piez et al., ed.) Elsevier, New York, N.Y., pp. 375–412(1984)).

Demineralized bone matrix (DBM) is a source of osteoinductive proteinsknown as bone morphogenetic proteins (BMP), and growth factors whichmodulate the proliferation of progenitor bone cells (see, e.g., Hauschkaet al., J. Biol. Chem. 261:12665–12674 (1986) and Canalis et al., J.Clin. Invest. 81:277–281 (1988)). Eight BMPs have now been identifiedand are abbreviated BMP-1 through BMP-8. BMP-3 and BMP-7 are also knownas osteogenin and osteogenic protein-1 (OP-1), respectively.

Unfortunately, DBM materials have little clinical use unless combinedwith particulate marrow autografts. There is a limit to the quantity ofDBM that can be surgically placed into a recipient's bone to produce atherapeutic effect. In addition, resorption has been reported to be atleast 49% (Toriumi et al., Arch. Otolaryngo. Head Neck Surg. 116:676–680(1990)).

DBM powder and osteogenin may be washed away by tissue fluids beforetheir osteoinductive potential is expressed. In addition, seepage oftissue fluids into DBM-packed bone cavities or soft-tissue collapse intothe wound bed are two factors that may significantly affect theosteoinductive properties of DBM and osteogenin. Soft-tissue collapseinto the wound bed may likewise inhibit the proper migration ofosteocompetent stem cells into the wound bed.

Human DBM in powder form is currently used by American dentists to packjaw bone cavities created during oral surgery. However, DBM in powderform is difficult to use.

Purified BMPs have osteoinductive effects in animals when delivered by avariety of means including FG (Hattori, T., Nippon. Seikeigeka. Gakkai.Zasshi. 64:824–834 (1990); Kawamura et al., Clin. Orthop. Rel. Res.235:302–310 (1988); Schlag et al., Clin. Orthop. Rel. Res. 227:269–285(1988) and Schwarz et al., Clin. Orthop. Rel. Res. 238:282–287 (1989))and whole blood clots (Wang et al., J. Cell. Biochem. 15F:Q20 Abstract(1990)). However, Schwarz et al. (supra.) demonstrated neither a clearpositive or negative effect of FG on ectopic osteoinduction orBMP-dependent osteoregeneration. Kawamura et al. (supra.) found asynergistic effect when partially purified BMP in FG was tested in anectopic non-bony site. Therefore, these results are inconsistent andconfusing.

TS also can serve as a “scaffold” which cells can use to move into awounded area to generate new tissues. However, commercially availablepreparations of FG and other TSs are too dense to allow cell migrationinto and through them. This limits their effectiveness in some in vivouses.

In one type of bone wound, called bone nonunion defects, there is aminimal gap above which no new bone formation occurs naturally.Clinically, the treatment for these situations is bone grafting.However, the source of bone autografts is usually limited and the use ofallogeneic bones involves a high risk of viral contamination. Because ofthis situation, the use of demineralized, virally inactivated bonepowder is an attractive solution.

D. Vascular Prostheses

Artificial vascular prostheses are frequently made out ofpolytetrafluoroethylene (PTFE) and are used to replace diseased bloodvessels in humans and other animals. To maximize patency rates andminimize the thrombogenicity of vascular prostheses various techniqueshave been used including seeding of nonautologous endothelial cells ontothe prothesis. Various substrates which adhere both to the vasculargraft and endothelial cells have been investigated as an intermediatesubstrate to increase endothelial cell seeding. These substrates includepreclotted blood (Herring et al., Surgery 84:498–504 (1978)), FG(Rosenman et al., J. Vasc. Surg. 2:778–784 (1985); Schrenk et al.,Thorac. Cardiovasc. Surg. 35:6–10 (1986); Köveker et al., Thorac.Cardiovasc. Surgeon 34: 49–51 (1986) and Zilla et al., Surgery105:515–522 (1989)), fibronectin (see, e.g., Kesler et al., J. Vasc.Surg. 3:58–64 (1986); Macarak et al., J. Cell Physiol. 116:76–86 (1983)and Ramalanjeona et al., J. Vasc. Surg. 3:264–272 (1986)), or collagen(Williams et al., J. Surg. Res. 38:618–629 (1985)). However, one generalproblem with these techniques is that nonautologous cells were used forthe seeding (see, e.g., Schrenk et al., supra) thus raising thepossibility of tissue rejection. In addition, a confluent endothelium isusually never established and requires months to do so if it is. As aresult of this delay, there is a high occlusion rate of vascularprostheses (see, e.g., Zilla et al., supra).

E. Angiogenesis

Angiogenesis is the induction of new blood vessels. Certain growthfactors such as HBGF-1 and HBGF-2 are angiogenic. However, their in vivoadministration attached to: collagen sponges (Thompson et al., Science241:1349–1352 (1988)); beads (Hayek et al., Biochem. Biophys. Res.Commun. 147:876–880 (1987)); solid PTFE fibers coated with collagenarranged in a sponge-like structure (Thompson et al., Proc. Natl. Acad.Sci. USA 86:7928–7932 (1989)); or by infusion (Puumala et al., BrainRes. 534:283–286 (1990)) resulted in the generation of random,disorganized blood vessels. These growth factors have not been usedsuccessfully to direct the growth of a new blood vessel(s) at a givensite in vivo. In addition, fibrin gels (0.5–10 mg/ml) implantedsubcutaneously in plexiglass chambers induce angiogenesis within 4 daysof implantation, compared to empty chambers, or chambers filled withsterile culture medium (Dvorak et al., Lab. Invest. 57:673 (1987)).

F. Site-Directed, Localized Drug Delivery

An efficacious, site-directed, drug delivery system is greatly needed inseveral areas of medicine. For example, localized drug delivery isneeded in the treatment of local infections, such as in periodontitis,where the systemic administration of antimicrobial agents isineffective. The problem after systemic administration usually lies inthe low concentration of the antimicrobial agent which can be achievedat the target site. To raise the local concentration a systemic doseincrease may be effective but also may produce toxicity, microbialresistance and drug incompatibility. To circumvent some of theseproblems, several alternative methods have been devised but none areideal. For example, collagen and/or fibrinogen dispersed in an aqueousmedium as an amorphous flowable mass, and a proteinaceous matrixcomposition which is capable of stable placement, have also been shownto locally deliver drugs (Luck et al., U.S. Reissue Patent 33,375; Lucket al., U.S. Pat. No. 4,978,332).

A variety of antibiotics (AB) have been reported to be released from FG,but only at relatively low concentrations and for relatively shortperiods of time ranging from a few hours to a few days (Kram et al., J.Surg. Res. 50:175–178 (1991)). Most of the ABs have been in freely watersoluble forms and have been added into the TS while it was beingprepared. However, the incorporation of tetracycline hydrochloridetetracycline hydrochloride (TET HCl) and other freely water solubleforms of ABs into FG has interfered with fibrin polymerization duringthe formation of the AB-supplemented FG (Schlag et al., Biomaterials4:29–32 (1983)). This interference limited the amount and concentrationof the TET HCl that could be achieved in the AB-FG mixture and appearedto be AB concentration dependent. The relatively short release time ofthe AB from the FG may reflect the relatively short life of theAB-supplemented TS or the form and/or quantity of the AB in the AB-TS.

G. Controlled Drug Release From TSs

For some clinical applications controlled, localized drug release isdesirable. As discussed above, some drugs, especially ABs, have beenincorporated into and been released from TSs such as FG. However, thereis little or no control over the duration of the drug release whichapparently is at least partially a reflection of the relatively shortlife of the drug-supplemented FG. Therefore, a means to stabilize FG andother TSs to allow for extended, localized drug release is desirable andneeded, as are new techniques for the incorporation and extended releaseof other supplements from TS.

H. The Disclosed TS Preparations Provide Life-Saving Emergency Treatmentfor Trauma Wounds

Despite continued advances in trauma care, a significant percentage ofthe population, both military and civilian, suffer fatal or severehemorrhage every year. An alarming number of fatalities are preventablesince the occur in the presence of those who could achieve life-savingcontrol of their wounds given adequate tools and training. Theavailability of the herein-disclosed TS satisfies the long-felt need fora advanced, easy-to-use, field-ready hemostatic preparation, to permitnot only trained medical personnel, but even untrained individuals torapidly reduce bleeding in trauma victims. Utilization of the disclosedTS preparations will result in a two-fold benefit: the reduction oftrauma death, and the decreased demand upon the available blood supply.

The disclosed technology would also be available for the treatment ofmassed casualties in disaster situation. When severe natural or man-madedisasters occur, local hospitals and clinics may be overwhelmed by thenumber of individuals requiring trauma care. Combined with the isolatingeffects of such disasters, the resulting demand for blood and bloodproducts often exceeds the locally available supplies. In many cases,the demand upon the local medical personnel also exceeds the availednumber of trained individuals. As a result, less seriously injuredpersons may be turned-away or given sub-optimal care. The availabilityof the easy-to-use, self-contained TS preparations disclosed below willpermit local medical personnel and disaster relief workers to providethe injured with temporary treatment until definitive care becomesavailable. Moreover, the disclosed TS preparations will permitself-treatment in disaster victims, until medical assistance can beprovided.

Often the only form of medical treatment that can be applied under suchcircumstances to prevent death due to blood loss is pressure dressings,tourniquets and pressure points. Unfortunately, however, each of thesetreatments requires continuous monitoring and attention. Since suchattention is not always possible in emergency or disaster situations,there is a clear need in the art for a simple, fast-acting, first-aidtreatment which can successfully control excessive blood loss.

The application of the disclosed TS preparations to the military isreadily apparent, particularly in isolated battlefield situations. Thesingle greatest cause of death on the battlefield is exsanguination,which until now has accounted for up to 50% of all combat casualties.

SUMMARY OF THE INVENTION

In one embodiment, this invention provides a composition of matter,comprising a TS, wherein the sealant does not inhibit full-thicknessskin wound healing.

In another embodiment, this invention provides a composition of matter,comprising: a TS, wherein the total protein concentration of the sealantis less than 30 mg/ml.

In another embodiment, this invention provides a composition of mattercomprising a supplemented TS wherein the total protein concentration isless than 30 mg/ml and the supplement is a growth factor(s) and/or adrug(s).

In another embodiment, this invention provides a composition of mattercomprising a supplemented TS wherein the total protein concentration isgreater than 30 mg/ml and the supplement is a growth factor(s) and/or adrug(s).

In another embodiment, this invention provides a composition of matterthat promotes the directed migration of animal cells, comprising: a TS;and an effective concentration of at least one growth factor, whereinthe concentration of the growth factor is effective in promoting thedirected migration of the animal cells.

In another embodiment, the present invention provides a composition ofmatter that promotes wound healing, comprising: a TS; and an effectiveconcentration of at least one growth factor, wherein the concentrationis effective in promoting wound healing.

In another embodiment, the present invention provides a composition ofmatter that promotes the endothelialization of a vascular prosthesis,comprising: a TS; and an effective concentration of at least one growthfactor, wherein the concentration is effective in promoting theendothelialization of a vascular prosthesis.

In another embodiment, the present invention provides a composition ofmatter that promotes the proliferation and/or differentiation of animalcells, comprising: a TS; and an effective concentration of at least onegrowth factor, wherein the concentration is effective in promotingproliferation and/or differentiation of animal cells.

In another embodiment, the present invention provides a composition ofmatter that promotes the localized delivery of at least one drug,comprising: a TS; and at least one drug.

In another embodiment, the present invention provides a composition ofmatter that promotes the localized delivery of at least one growthfactor, comprising: a TS; and at least one growth factor.

In another embodiment, the present invention provides a process forpromoting the healing of wounds, comprising applying to the wound, acomposition that contains a TS and an effective concentration of atleast one growth factor, wherein the concentration is effective topromote wound healing.

In another embodiment, the present invention provides a process forpromoting the endothelialization of a vascular prosthesis, comprisingapplying to the vascular prosthesis a composition that contains a TS andan effective concentration of at least one growth factor, wherein theconcentration is effective to promote the endothelialization of avascular prothesis.

In another embodiment, the present invention provides a process forpromoting the proliferation and/or differentiation of animal cells,comprising placing the cells in sufficient proximity to a TS whichcontains an effective concentration of at least one growth factor,wherein the concentration is effective in promoting the proliferationand/or differentiation of the cells.

In a further embodiment, the present invention provides a process forthe localized delivery of at least one drug to a tissue, comprisingapplying to the tissue a TS which contains at least one drug.

In another embodiment, the present invention provides a process for thelocalized delivery of at least one growth factor to a tissue, comprisingapplying to the tissue a TS which contains at least one growth factor.

In another embodiment, this invention provides a process for producingthe directed migration of animal cells, comprising: placing insufficient proximity to the cells, a TS which contains an effectiveconcentration of at least one growth factor, wherein the concentrationis effective to produce the desired directed migration of said cells.

In another embodiment, this invention provides a simple to use, fastacting, field-ready fibrin bandage for applying a tissue sealingcomposition to wounded tissue in a patient, comprising an occlusivebacking, affixed to which is a layer of dry materials comprising aneffective amount, in combination, of (a) dry, virally-inactivated,purified fibrinogen, (b) dry, virally-inactivated, purified thrombin,and as necessary (c) effective amounts of calcium and/or Factor XIII toproduce a tissue-sealing fibrin clot upon hydration.

In a further embodiment, this invention provides a method of treatingwounded tissue in a patient by applying to said wound a fibrin bandage,comprising: (1) a occlusive backing, affixed to which is a layer of drymaterials comprising an effective amount, in combination, of (a) dry,virally-inactivated, purified fibrinogen, (b) dry, virally-inactivated,purified thrombin, and as necessary (c) effective amounts of calciumand/or Factor XIII to produce a tissue-sealing fibrin clot uponhydration.

In yet another embodiment, this invention provides a simple to use, fastacting, field-ready fibrin dressing for treating wounded tissue in apatient, is formulated as an expandable foam comprising an effectiveamount, in combination, of (1) virally-inactivated, purified fibrinogen,(2) virally-inactivated, purified thrombin, and as necessary (3) calciumand/or Factor XIII; wherein said composition does not significantlyinhibit full-thickness skin wound healing.

While in a further embodiment, this invention provides a method oftreating wounded tissue in a patient by applying to said wound a tissuesealant expandable foam dressing, comprising an effective amount, incombination, of (1) virally-inactivated, purified fibrinogen, (2)virally-inactivated, purified thrombin, and as necessary (3) calciumand/or Factor XIII; wherein said composition does not significantlyinhibit full-thickness skin wound healing.

In the embodiments of this invention, the TS may be FG.

In the various embodiments of the invention FG may be made from themixing of topical fibrinogen complex (TFC), human thrombin and calciumchloride. Varying the concentration of the TFC has the most significanteffect upon the density of the final FG matrix. Varying theconcentration of the thrombin has an insignificant effect upon the totalprotein concentration of the final FG, but has a profound effect uponthe time required for the polymerization of the fibrinogen component ofthe TFC into fibrin. While this effect is well known, it is notgenerally appreciated that it may be used to maximize the effectivenessof the FG, when it is used alone or supplemented. Because of this effectone can alter the time between the mixing of the FG components and thesetting of the FG. Thus, one can allow the FG to flow more freely intodeep crevices in a wound, permitting it to fill the wound completelybefore the FG sets. Alternatively, one can allow the FG to set quicklyenough to prevent it from exiting the wound site, especially if thewound is leaking fluid under pressure (i.e., blood, lymph, intercellularfluid, etc). This property is also important to keep the FG fromclogging delivery devices with long passages, i.e., catheters,endoscopes, etc., which is important to allow the application of the FGor supplemented FG to sites in the body that are only accessible bysurgery. This effect is also important in keeping the insolublesupplements in suspension and preventing them from settling in theapplicator or in the tissue site.

As used herein, TFC is a lyophilized mixture of human plasma proteinswhich have been purified and virally inactivated. When reconstituted TFCcontains:

Total Protein: 100–130 mg/ml Fibrinogen: (as clottable protein) 80% oftotal protein (minimum) Albumin (Human): 5–25 mg/ml Plasminogen: 5 mg/mlFactor XIII: 10–40 Units/ml Polysorbate-80: 0.3% (maximum) pH: 7.1–7.5.

The reconstituted TFC may also contain trace amounts of fibronectin.

As used herein, human thrombin is a lyophilized mixture of human plasmaproteins, which have been purified and virally inactivated. Whenreconstituted it contains:

Thrombin Potency: 300 ± 50  International Units/ml Albumin (Human): 5mg/ml Glycine: 0.3 M ± .05 M pH: 6.5–7.1.

Calcium chloride is added in sufficient concentration to activate thethrombin. As long as there is sufficient calcium, its concentration isnot important.

In the compositions of this invention containing a growth factor, thecomposition may contain an inhibiting compound(s) and/or potentiatingcompound(s), wherein the inhibiting compound(s) inhibit the activitiesof the sealant that interfere with any of the biological activities ofthe growth factor, the potentiating compound(s) potentiate, mediate orenhance any of the biological activities of the growth factor, andwherein the concentration of the inhibiting or potentiating compound iseffective for achieving the inhibition, potentiation, mediation orenhancement.

The growth factor-supplemented TSs of this invention are useful forpromoting the healing of wounds, especially those that do not readilyheal, such as skin ulcers in diabetic individuals, and for deliveringgrowth factors including, but not limited to, FGF-1, FGF-2, FGF-4,PDGFs, EGFs, IGFs, PDGF-bb, BMP-1, BMP-2, OP-1, TGF-β,cartilage-inducing factor-A (CIF-A), cartilage-inducing factor-B(CIF-B), osteoid-inducing factor (OIF), angiogenin(s), endothelins,hepatocyte growth factor and keratinocyte growth factor, and providing amedium for prolonged contact between a wound site and the growthfactor(s). The growth factor-supplemented TS may be used to treat burnsand other skin wounds and may comprise a TS and, in addition to thegrowth factor(s), an antibiotic(s) and/or an analgesic(s), etc. Thegrowth factor-supplemented TS may be used to aid in the engraftment of anatural or artificial graft, such as skin to a skin wound. They may alsobe used cosmetically, for example in hair transplants, where the TSmight contain FGF, EGF, antibiotics and minoxidil, as well as othercompounds. An additional cosmetic use for the compositions of thisinvention is to treat wrinkles and scars instead of using silicone orother compounds to do so. In this embodiment, for example, the TS maycontain FGF-1, FGF-4, and/or PDGFs, and fat cells. The growthfactor-supplemented TSs may be applied to surgical wounds, broken bonesor gastric ulcers and other such internal wounds in order to promotehealing thereof. The TSs of this invention may be used to aid theintegration of a graft, whether artificial or natural, into an animal'sbody as for example when the graft is composed of natural tissue. TheTSs of this invention can be used to combat some of the major problemsassociated with certain conditions such as periodontitis, namelypersistent infection, bone resorption, loss of ligaments and prematurere-epithelialization of the dental pocket.

In another embodiment, this invention provides a mixture of FG, DBMand/or purified BMP's. This mixture provides a matrix that allows thecellular components of the body to migrate into it and thus produceosteoinduction where needed. The matrix composition in terms of proteins(such as fibrinogen and Factor XIII), enzymes (such as thrombin andplasmin), BMPs, growth factors and DBM and their concentrations areadequately formulated to optimize the longevity of this temporalscaffolding structure and the osteoinduction which needs to occur. Allthe FG components are biodegradable but during osteogenesis the mixtureprovides a non-collapsible scaffold that can determine the shape andlocation of the newly formed bone. Soft tissue collapse into the bonynonunion defect, which is a problem in bone reconstructive surgery, willthus be avoided. The use of TS supplemented with growth factors such asCIF-A and CIF-B, infra, which promote cartilage development, will beuseful in the reconstruction of lost or damaged cartilage and/or damagedbone.

In a preferred embodiment, an effective concentration of HBGF-1 is addedto a FG in order to provide a growth factor-supplemented TS thatpossesses the ability to promote wound healing. In another preferredembodiment, an effective amount of a platelet-derived extract is addedto a FG. In other preferred embodiments, an effective concentration of amixture of at least two growth factors are added to FG and an effectiveamount of the growth factor(s)-supplemented FG is applied to the woundedtissue.

In addition to growth factors, drugs, polyclonal and monoclonalantibodies and other compounds, including, but not limited to, DBM andBMPs may be added to the TS. They accelerate wound healing, combatinfection, neoplasia, and/or other disease processes, mediate or enhancethe activity of the growth factor in the TS, and/or interfere with TScomponents which inhibit the activities of the growth factor in the TS.These drugs may include, but are not limited to: antibiotics, such astetracycline and ciprofloxacin; antiproliferative/cytotoxic drugs, suchas 5-fluorouracil (5-FU), taxol and/or taxotere; antivirals, such asgangcyclovir, zidovudine, amantidine, vidarabine, ribaravin,trifluridine, acyclovir, dideoxyuridine and antibodies to viralcomponents or gene products; cytokines, such as α- or β- orγ-Interferon, α- or β-tumor necrosis factor, and interleukins; colonystimulating factors; erythropoietin; antifungals, such as diflucan,ketaconizole and nystatin; antiparasitic agents, such as pentamidine;anti-inflammatory agents, such as α-1-anti-trypsin andα-1-antichymotrypsin; steroids; anesthetics; analgesics; and hormones.Other compounds which may be added to the TS include, but are notlimited to: vitamins and other nutritional supplements; hormones;glycoproteins; fibronectin; peptides and proteins; carbohydrates (bothsimple and/or complex); proteoglycans; antiangiogenins; antigens;oligonucleotides (sense and/or antisense DNA and/or RNA); BMPs; DBM;antibodies (for example, to infectious agents, tumors, drugs orhormones); and gene therapy reagents. Genetically altered cells and/orother cells may also be included in the TSs of this invention. Theosteoinductive compounds which can be used in practicing this inventioninclude, but are not limited to: osteogenin (BMP3); BMP-2; OP-1; BMP-2A,-2B, and -7; TGF-β, HBGF-1 and -2; and FGF-1 and -4. In addition,anything which does not destroy the TS can be added to the TSs of thisinvention.

The studies reported herein unexpectedly demonstrate that the inclusionof compounds such as the free base TET or ciprofloxacin (CIP) HCl, in FGor the treatment of FG therewith confers extended longevity to thesupplemented FG. This phenomenon can be exploited to increase theduration of a drug's release from the TS. Alternatively, this phenomenoncan be exploited to modulate the release of another drug(s) other thanthe compound used to stabilize the FG, which is (are) also incorporatedinto the TET-FG, and/or to cause the FG to persist for a greater periodin vivo or in vitro.

In general, poorly water soluble forms of a drug, such as the free baseof TET, increase the delivery of the drug from the TS more than freelywater soluble forms thereof. Therefore, the drug may be bound to aninsoluble carrier, such as fibrinogen or activated charcoal, within theTS to prolong the delivery of the drug from the supplemented TS.

In another embodiment, the supplemented TS can be used in organoids andcould contain, for example, growth factors such as FGF-1, FGF-2, FGF-4and OP-1.

In another embodiment, this invention provides a composition thatpromotes the localized delivery of a poorly water soluble form of anantibiotic(s), such as the free base form of TET, and other drug(s),comprising a TS and an effective concentration of at least one poorlywater soluble form of an antibiotic. Similar delivery methods are alsoapplied to other drugs, antibodies, oligonucleotides, cytotoxins, cellproliferation inhibitors, osteogenic or cartilage inducing compounds,growth factors or other supplements herein disclosed.

The present invention has several advantages over the previously used TScompositions and methods. The first advantage is that the growth factor-and/or drug-supplemented TSs of the present invention have many of thecharacteristics of an ideal biodegradable carrier, namely: they can beformulated to contain only human proteins thus eliminating or minimizingimmunogenicity problems and foreign-body reactions; their administrationis versatile; and their removal from the host's tissues is not requiredbecause they are degraded by the host's own natural fibrinolytic system.

A second advantage is that the present invention provides a good way toeffectively deliver growth factors and/or drugs for a prolonged periodof time to an internal or external wound. It appears that some growthfactor receptors must be occupied for at least 12 hours to produce amaximal biological effect. Previously, there was no way to do this. Thepresent invention allows for prolonged contact between the growth factorand its receptors to occur, and thus allows for the production of strongbiological effects.

A third advantage of the present invention is that animal cells canmigrate into and through, and grow in the TSs of the present invention.This aids engraftment of the cells to neighboring tissues andprostheses. Based on the composition of the TSs which are available inEurope, it is expected that this is not possible with theseformulations. Instead, animal cells must migrate around or digestcommercially available TS. Since the importation into the U.S. ofcommercially available TSs from Europe is illegal (their use in the USAhas not been approved by the U.S. FDA).

A fourth advantage is that because of its initial liquid nature, the TSof the present invention can cover surfaces more thoroughly andcompletely than many previously available delivery systems. This isespecially important for the use of the present invention in coatingbiomaterials and in the endothelialization of vascular prosthesesbecause the growth factor-supplemented FG will coat the interior,exterior and pores of the vascular prosthesis. As a result of this, plusthe ability of endothelial cells to migrate into and through the TS,engraftment of autologous endothelial cells will occur along the wholelength of the vascular prosthesis, thereby decreasing itsthrombogenicity and antigenicity. With previously used TSs, engraftmentstarted at the ends of the vascular prosthesis and proceeded, if at all,into the interior of the same, thus allowing a longer period forthrombogenicity and antigenicity to develop. Previously used TSs forvascular prostheses also primarily were seeded with nonautologous cellswhich could be rejected by the body and could be easily washed off bythe shearing force of blood passing through the prosthesis.

A fifth advantage is that the supplemented and unsupplemented TS of thisinvention can be molded and thus can be custom made into almost anydesired shape. For example, TS such as FG can be supplemented with BMPsand/or DBM and can be custom made into the needed shape to mostappropriately treat a bone wound. This cannot be done with DBM powderalone because DBM powder will not maintain its shape.

A sixth advantage is that the AB-supplemented FG of this invention, suchas TET-FG, has unexpectedly increased the longevity and stability of theFG compared to that of the unsupplemented FG. This increased stabilitycontinues even after appreciable quantities of the AB are no longerremaining in the FG. For example, soaking a newly formed FG clot in asaturated solution of TET produced from free base TET, or in a solutionof CIP HCl, produces a FG clot which is stable and preserved even aftersubstantially all the TET or CIP has left the FG clot. While not wishingto be bound by any theory as to how this effect is produced, it isbelieved that the AB, such as TET or CIP, inhibits plasminogen which isin the TFC and breaks down the FG. Once the plasminogen is inhibited,its continued inhibition does not appear to depend on appreciablequantities of the TET or CIP remaining in the FG. As a result of thisstabilizing effect, one can expect an increased storage shelf life ofthe TS, and possibly an increased persistence in vivo.

The seventh advantage of the present invention is a direct result of theprolonged longevity and stability of the TS. As a result of thisunexpected increase in stability of the TS, AB-supplemented FG can beused to produce localized, long term delivery of a drug(s) and/or agrowth factor(s). This delivery will continue even after the stabilizingdrug, such as TET or CIP, has substantially left the TS. Inclusion of asolid form, preferably a poorly water soluble form of a drug such asfree base, into a TS that has been stabilized by, for example, TET orCIP, then allows the stabilized TS to deliver that drug (or growthfactor) locally for an extended period of time. Some forms of drugs,such as free base TET, allow for both stabilization of the TS and forprolonged drug delivery. Other drugs may do one or the other but notboth. A compound used for the stabilization of a TS to produceprolonged, localized drug delivery is not previously known in the art.

An eighth advantage of the present invention is that it allowssite-directed angiogenesis to occur in vivo. While others havedemonstrated localized non-specific angiogenesis, supra, no one else hasused a TS to promote site-directed angiogenesis.

A ninth advantage of the present invention is that because thecomponents of the TS can be formulated into several forms of simple touse, fast-acting field dressings, it is now possible to control bleedingfrom hemorrhaging trauma wounds, thereby saving numerous lives thatpreviously would have been lost. Although life-saving methods oftreating such wounds are possible by trained medical personal or infully-equipped clinics and hospitals, the present invention satisfiessociety's long-felt need for an easy-to-use, first-aid (or evenself-applied) treatment that will, in emergency or disaster situations,allow an untrained individual to treat traumatic injuries to controlhemorrhage until medical assistance is available.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A–1F show Western blots of gels on which samples containingHBGF-1β had been incubated with 250 U/ml thrombin in the presence ofincreasing concentrations of heparin. Solutions containing HBGF-1β (10μg/ml), thrombin (250 μg/ml), and increasing concentrations of heparin(0, 0.5, 5, 10, 20, and 50 units/ml) were incubated at 37° C. for 72hours. Aliquots were periodically removed from each of the incubatingmixtures and were loaded onto 8% SDS polyacrylamide gels that wereprepared and run as described by Laemmli (Nature 227:680 (1970)). Thegel was then electroblotted onto nitrocellulose and the bandcorresponding to HBGF-1β was identified using an affinity-purifiedpolyclonal rabbit antiserum to HBGF-1β.

The concentrations of heparin in the incubating mixtures were: panel A)0 units/ml (u/ml); panel B) 0.5 u/ml; Panel C) 5 u/ml; panel D) 10 u/ml;panel E) 20 u/ml; and panel F) 50 u/ml. In the gels pictured in each ofpanels A–F, each lane contains the following: lane 1 contains SDS-PAGElow molecular weight standards; lane 2 contains biotinylated standards;lane 3 contains 10 μg/ml HBGF-1β; lane 4 contains 250 u/ml thrombin; andlanes 5–13 contain samples removed from the incubating mixtures at times0, 1, 2, 4, 6, 8, 24, 48, and 72 hours.

FIG. 2 shows the incorporation of ³H-thymidine as a function of relativeHBGF-1β concentration. Samples of the HBGF-1β were incubated, asdescribed in FIG. 1 and Example 2, in the presence of 250 U/ml thrombinand 5 U/ml heparin for 0, 24 or 72 hours. Dilutions of these sampleswere then added to NIH 3T3 cells, which were plated as described inExample 3. CPM is plotted v. HBGF-1β concentration.

FIG. 3. Typical pattern of human umbilical vein endothelial cells after7 days' growth on FG supplemented with 100 ng/ml of active, wild-typeFGF-1. Note the large number of cells and their elongated shape. Comparewith the paucity of cells grown on unsupplemented FG (FIG. 5).

FIG. 4. Typical pattern of human umbilical vein endothelial cells after7 days' growth on FG supplemented with 10 ng/ml of active, wild-typeFGF-1. Note the large number of cells and their elongated shape. Comparewith the paucity of cells grown on unsupplemented FG (FIG. 5).

FIG. 5. Typical pattern of human umbilical vein endothelial cells after7 days' growth on unsupplemented FG. Note the small number of cells,compared to the number of cells in FIGS. 3 and 4, which indicates aslower proliferation rate.

FIG. 6. Typical pattern of human umbilical vein endothelial cells after7 days' growth on FG supplemented with 100 ng/ml of inactive, mutantFGF-1. Note the small number of cells, compared to the number of cellsin FIGS. 3 and 4, which indicates a slower proliferation rate.

FIG. 7. Typical pattern of human umbilical endothelial cells 24 hoursafter having been embedded in FG at a concentration of 10⁵ cells per mlof FG. The protein and thrombin concentrations of the FG were 4 mg/mland 0.6 NIH units/ml, respectively. Note, their elongated, multipodialmorphology and that they formed a cellular network where they came incontact with each other. Compare with the cobblestone shape of similarcells grown in fibronectin (FIG. 9.)

FIG. 8. Typical pattern of human umbilical endothelial cells 48 hoursafter having been embedded in FG at a concentration of 10⁵ cells per mlof FG. The culture conditions were as described in FIG. 7. Note thefurther accentuated, elongated and multipodial morphology and increasingdevelopment of cellular networks. Compare with the cobblestone shapedcells grown in fibronectin (FIG. 10) and note the lack of a cellularnetwork in the latter.

FIG. 9. Typical pattern of human umbilical endothelial cells 24 hoursafter having been cultured on a surface coated with fibronectin. Notethe cobblestone shape of the cells and the lack of cellular networks.Compare to FIG. 7.

FIG. 10. Typical pattern of human umbilical endothelial cells 48 hoursafter having been cultured in a commonly used film of fibronectin. Notethe cobblestone shape of the cells and the lack of cellular networks.Compare to FIG. 8.

FIG. 11. Micrographs of cross sections of PTFE vascular grafts that wereexplanted from dogs after 7 days (panels A, C, E) or 28 days (panels B,D, F). Prior to implantation, the grafts were either untreated (A andB), coated with FG alone (C and D), or coated with FG supplemented withheparin and HBGF-1 (E and F).

Untreated controls (A & B) showed minimal mesenchymal tissue ingrowth,with both their interstices filled with, and their luminal surfacescoated with fibrin coagulum. The FG-treated grafts showed mesenchymaltissue ingrowth in only the outer half of the grafts' interstices, withthe rest being filled with fibrin coagulum. Very few interstitialcapillaries were present. In contrast, the grafts treated with FGcontaining FGF-1 showed more abundant interstitial ingrowth and by 28days showed numerous capillaries, myofibroblasts and macrophages, withinner capsules consisting of several layers of myofibroblasts beneathconfluent endothelial cell layers. Results of similar grafts after 128days of implantation were similar, with greater numbers of capillariesin the FG+FGF-1 group (data not shown).

FIG. 12. Scanning electron micrographs of the inner lining of thevascular grafts described in FIG. 11 after 28 days of implantation. Thegrafts were either untreated (G), coated with FG alone (H), or coatedwith FG supplemented with heparin and HBGF-1 (I). Untreated controlgrafts (G) showed sparse areas of endothelial cell coverage amidst areasof thrombus containing red blood cells, platelets, and areas of exposedPTFE graft material (visible in the center and top of the picture).Grafts coated with FG alone (H) showed islands of endothelial cellsamidst areas of fibrin coagulum. In contrast, grafts treated withFG+HBGF-1 (I) showed confluent endothelial cells oriented along thedirection of blood flow.

FIG. 13. Graph showing the inhibition of smooth muscle cellproliferation by the release of tributyrin from supplemented fibrinsealant. Unsupplemented fibrin sealant=(□); tributyrin-supplementedfibrin sealant=(▪).

FIG. 14. Preparation of disc-shaped implants 1 mm thick and 8 mm indiameter prepared using an aluminum mold.

FIG. 15. Diagram illustrating intramuscular bioassay for the inductionof bone formation in rats by DBM alone, by FG implants or by DBM-FG.

FIG. 16. Diagram illustrating the induction of bone formation incalvarial implants by DBM-FG.

FIG. 17. Radio-opacity data at 28 days postoperative from intramuscularimplants of DBM-FG, DBM or FG.

FIG. 18. Radio-opacity data from DBM-FG (30 mg/ml) calvarial implants at28 days, 3 months and 4 months postoperative.

FIG. 19. FIG. 19A is a photograph of a craniotomy site at 28 days postsurgery in a treated animal. FIG. 19B is a photograph of the calvarialwound from an untreated control at 28 days postoperative. Note that onlyfibrous connective tissue has developed across the craniotomy wound.

FIG. 20. Photograph from the craniotomy wounds of animals which weretreated with DBM particles only.

FIG. 21. Photograph of new bone formed in the craniotomy site inresponse to DBM-FG (15 mg/ml).

FIG. 22. Photograph of new bone formed in the craniotomy site inresponse to DBM-FG (15 mg/ml). Note that typically more bone marrowformed in craniotomy wounds that had been implanted with DBM-FG disksthan with DBM implants alone.

FIG. 23. The release of TET from 3×6 mm diameter disks of FG at 37° C.The concentration of the released TET was measuredspectrophotometrically in 2 ml of PBS supernatant that had been replaceddaily. Two of these “static” in vitro experiments were carried out withidentical results. The results of one of them is shown here.

FIG. 24. The release of TET from 3×6 mm diameter disks of FG at 37° C.The disks contained 100 mg/ml of TET and were placed in closed vesselsfilled with 2 ml of PBS. The TET concentration was measuredspectrophotometrically in the PBS effluent which had been continuouslyexchanged at a rate of 3 ml/day. The volume of the PBS supernatant hadbeen kept constant at approximately 2 ml. The data are the average oftwo experiments.

FIG. 25. The release of TET into saliva from 3×6 mm diameter diskscontaining 50 or 100 TET mg/ml FG at 37° C. The TET concentration wasmeasured spectrophotometrically in 0.75 ml of saliva supernatant thathad been replaced daily. The saliva used in these experiments had beenpooled from ten donors, centrifuged, filtered and kept at 4° C.

FIG. 26. The stability of TET-supplemented FG was increased compared tothat of control FG. Photographs of 3×6 mm diameter FG matrixes withoutTET and with 50 and 100 mg/ml TET over a period of 15 days. The diskshad been kept in 0.75 ml of saliva which had been changed daily. Thesaliva had been pooled from 10 donors. It had then been centrifuged,filtered and stored at 4° C. before use in this experiment. Note that atnine days, the FG matrix which did not contain TET had decayed more thanthe matrices which contained either 50 or 100 mg/ml of TET Also notethat at 15 days, the FG matrix which did not contain TET had almosttotally decayed, whereas the FG matrices which contained 50 or 100 mg/mlof TET were almost unchanged. Therefore, the inclusion of 50 or 100mg/ml of TET dramatically prolonged the longevity of FG matrices insaliva in vitro.

FIG. 27. Antibacterial activity of TET released from TET-supplementedFG. Two ml PBS surrounding the 3×6 mm TET-supplemented FG disks wasreplaced daily. For testing the antimicrobial activity of the releasedTET, 6 mm paper disks impregnated with the collected eluates wereincubated for 18 hours at 37° C. on agar plates containing E. coli. Thenthe diameter of the zone of inhibition was measured.

FIG. 28. The release of ciprofloxacin, amoxicillin and metronidazolefrom FG matrices. Individual 3×6 mm diameter disks containing 100 mg/mlof the respective antibiotics were immersed in 2 ml ofphosphate-buffered saline at 37° C. The supernatant was replaced dailyand the antibiotic concentration was measured spectrophotometrically at275, 274 and 320 nm, respectively.

FIG. 29. The release of TET from TET-supplemented FG disks wasproportional to the temperature of the PBS bathing the TET-FG disks.

FIG. 30. The effect of FG protein concentration on the release of TETfrom TET-FG. Note that higher FG protein concentrations resulted in aslower TET release rate from the TET-FG.

FIG. 31A. Graph showing the elution profile of in vitro release ofantibiotic from a supplemented fibrin sealant disks.

FIG. 31B. Graph showing the elution profile of in vitro vs. in vivorelease of tetracycline from supplemented fibrin sealant disks.

FIG. 31C. Graph showing the inhibition of bacterial growth bytetracycline supplemented fibrin sealant disks as compared tounsupplemented fibrin sealant disks and culture media alone.

FIG. 32. The release of 5-FU from 5-FU-supplemented FG was prolonged bythe use of solid forms of 5-FU.

FIG. 33. Graph showing the effect over time of supernatants fromtaxol-supplemented fibrin sealant composition on rapidly proliferatinghuman ovarian carcinoma cells (OVCAR).

FIG. 34. Dose-response relationship of the chemotactic response of NIH3T3 fibroblasts to Fibronectin. A step gradient of increasingconcentrations of Fibronectin was added to the lower wells of themodified Boyden's chambers. The data are expressed as means +/−S.E. ofmigrated cells per high power field and demonstrate that, as a functionof dose, fibronectin induced the chemotaxis of NIH 3T3 cells toward it.

FIG. 35. Dose-response relationship of the chemotactic response of NIH3T3 fibroblasts to FGF-1. A step gradient of increasing concentrationsof FGF-1 was added to the lower wells of the modified Boyden's chambersin the presence of heparin. The data are expressed as the means +/−S.E.of migrated cells per high power field and demonstrate that, as afunction of dose, FGF-1 induced the chemotaxis of fibroblasts toward it.

FIG. 36. Dose-response relationship of the chemotactic response of NIH3T3 fibroblasts to FGF-2. A step gradient of increasing concentrationsof FGF-2 was added to the lower wells of the modified Boyden's chambers.The data are expressed as the means +/−S.E. of migrated cells per highpower field and demonstrate that, as a function of dose, FGF-2 inducedthe chemotaxis of fibroblasts toward it.

FIG. 37. Dose-response relationship of the chemotactic response of NIH3T3 fibroblasts to FGF-4. A step gradient of increasing concentrationsof FGF-4 was added to the lower wells of the modified Boyden's chambersin the presence of heparin. The data are expressed as the means +/−S.E.of migrated cells per high power field and demonstrate that, as afunction of dose, FGF-4 induced the chemotaxis of fibroblasts toward it.

FIG. 38. Dose-response relationship of the chemotactic response of humandermal fibroblasts (HDFs) to FGF-1. A step gradient of increasingconcentrations of FGF-1 was added to the lower wells of the modifiedBoyden's chambers in the presence of heparin. The data are expressed asthe means +/−S.E. of migrated cells per high power field and demonstratethat, as a function of dose, FGF-1 induced the chemotaxis of HDFs towardit.

FIG. 39. Dose-response relationship of the chemotactic response of HDFsto FGF-2. A step gradient of increasing concentrations of FGF-2 wasadded to the lower wells of the modified Boyden's chambers. The data areexpressed as the means +/−S.E. of migrated cells per high power fieldand demonstrate that, as a function of dose, FGF-2 induced thechemotaxis of HDFs toward it.

FIG. 40. Dose-response relationship of the chemotactic response of HDFsto FGF-4. A step gradient of increasing concentrations of FGF-4 wasadded to the lower wells of the modified Boyden's chambers. The data areexpressed as the means +/−S.E. of migrated cells per high power fieldand demonstrate that, as a function of dose, FGF-4 induced thechemotaxis of HDFs toward it.

FIG. 41. Dose-response relationship of the chemotactic response of HDFsto FGF-4 in solution and in FG. FGF-4 was incorporated into FG andplaced in the bottom well of chemotaxis chambers. The amount of FGF inthe FG was sufficient to result in the indicated concentrations whenevenly distributed throughout the FG and medium in the lower chamber.Negative controls included medium alone and FG without FGF. Mediumcontaining FGF-4 at a concentration of 10 ng/ml in the lower chamber wasutilized as a positive control. The data are expressed as the means+/−S.E. of migrated cells per high power field and demonstrate that, asa function of dose, FGF-4 released from FG induced the chemotaxis ofHDFs toward the FG.

FIG. 42. Diagram of a self-contained TS Wound Dressing.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which this invention belongs. All patents and publicationsmentioned herein are incorporated by reference.

As used herein, a wound includes damage to any tissue in a livingorganism. The tissue may be an internal tissue, such as the stomachlining or a bone, or an external tissue, such as the skin. As such awound may include, but is not limited to, a gastrointestinal tractulcer, a broken bone, a neoplasia, and cut or abraided skin. A wound maybe in a soft tissue, such as the spleen, or in a hard tissue, such asbone. The wound may have been caused by any agent, including traumaticinjury, infection or surgical intervention.

As used herein, TS is a substance or composition that, upon applicationto a wound, seals the wound, thereby reducing blood loss and maintaininghemostasis. As used herein, FG is a composition, prepared fromrecombinant or plasma proteins, that upon application to a wound forms aclot, thereby sealing the wound, reducing blood loss and maintaininghemostasis. FG, supra, is a form of TS.

As used herein, supplemented TS includes any TS that, withoutsubstantial modification, can serve as a carrier vehicle for thedelivery of a growth factor, drug or other compound, or mixturesthereof, and that, by virtue of its adhesive or adsorptive properties,can maintain contact with the site for a time sufficient for thesupplemented TS to produce its desired effect, for example to promotewound healing.

As used herein, a growth factor-supplemented TS is a TS to which atleast one growth factor has been added at a concentration that iseffective for its stated purpose. The growth factor can, for example,accelerate, promote or improve wound healing, or tissue (re)generation.The growth factor-supplemented TSs may also contain additionalcomponents, including drugs, antibodies, anticoagulants and othercompounds that: 1) potentiate, stimulate or mediate the biologicalactivity of the growth factor(s) in the TS; 2) decrease the activitiesof components of the growth factor-supplemented TS which would inhibitor destroy the biological activities of the growth factor(s) in thesealant; or 3) allow prolonged delivery of the supplement from the TS;4) possess other desirable properties.

As used herein, a potentiating compound is a compound that mediates orotherwise increases the biological activity of a growth factor in theTS. Heparin is an example of a compound that potentiates the biologicalactivity of HBGF-1.

As used herein, an inhibiting compound is a compound that inhibits,interferes with, or otherwise destroys a deleterious activity of acomponent of the TS that would interfere with or inhibit the biologicalactivity of a growth factor or factors in the TS. Inhibiting compoundsmay exert their effect by protecting the growth factor from degradation.An inhibiting compound does not, however, inhibit any activities thatare essential for the desired properties, such as, for example, woundhealing of the growth factor-supplemented TS. An example of aninhibiting compound is heparin.

As used herein, a growth factor includes any soluble factor thatregulates or mediates cell proliferation, cell differentiation, tissueregeneration, cell attraction, wound repair and/or any developmental orproliferative process. The growth factor may be produced by anyappropriate means including extraction from natural sources, productionthrough synthetic chemistry, production through the use of recombinantDNA techniques and any other techniques, including virally inactivated,growth factor(s)-rich platelet releasate, which are known to those ofskill in the art. The term growth factor is meant to include anyprecursors, mutants, derivatives, or other forms thereof which possesssimilar biological activity(ies), or a subset thereof, to those of thegrowth factor from which it is derived or otherwise related.

As used herein, HBGF-1, which is also known to those of skill in the artby alternative names, such as endothelial cell growth factor (ECGF) andFGF-1, refers to any biologically active form of HBGF-1, includingHBGF-1β, which is the precursor of HBGF-1α and other truncated forms,such as FGF. U.S. Pat. No. 4,868,113 to Jaye et al., herein incorporatedby reference, sets forth the amino acid sequences of each form of HBGF.HBGF-1 thus includes any biologically active peptide, includingprecursors, truncated or other modified forms, or mutants thereof thatexhibit the biological activities, or a subset thereof, of HBGF-1.

Other growth factors may also be known to those of skill in the art byalternative nomenclature. Accordingly, reference herein to a particulargrowth factor by one name also includes any other names by which thefactor is known to those of skill in the art and also includes anybiologically active derivatives or precursors, truncated mutant, orotherwise modified forms thereof.

As used herein, biological activity refers to one or all of theactivities that are associated with a particular growth factor in vivoand/or in vitro. Generally, a growth factor exhibits several activities,including mitogenic activity (the ability to induce or sustain cellularproliferation) and also non-mitogenic activities, including the abilityto induce or sustain differentiation and/or development. In addition,growth factors are able to recruit or attract particular cells fromwhich the proliferative and developmental processes proceed. Forexample, under appropriate conditions HBGF-1 can recruit endothelialcells and direct the formation of vessels therefrom. By virtue of thisactivity, growth factor-supplemented TS may thereby provide a means toenhance blood flow and nutrients to specific sites.

As used herein, extended longevity means at least a two fold increase inthe visually observable, useful in vitro lifespan of a TS.

As used herein, demineralized bone matrix (DBM) means the organic matrixof bone that remains after bone is decalcified with hydrochloric oranother acid.

As used herein, bone morphogenetic proteins (BMPs) mean a group ofrelated proteins originally identified by their presence inbone-inductive extracts of DBM. At least 8 related members have beenidentified and are designated BMP-1 through BMP-8. The BMPs are alsoknown by other names. BMP-2 is also known as BMP-2A. BMP-4 is also knownas BMP-2B. BMP-3 is also known as osteogenin. BMP-6 is also known asVgr-1. BMP-7 is also known as OP-1. Bone morphogenetic proteins is meantto include, but is not limited to BMP-1 through BMP-8.

As used herein, augmentation means using a supplemented orunsupplemented TS to change the internal or external surface contour ofa component of an animal's body.

As used herein, a damaged bone is a bone which is broken, fractured,missing a portion thereof, or otherwise not healthy, normal bone.

As used herein, a deficient bone is a bone which has an inadequate shapeor volume to perform its function.

As used herein, bone or DBM which is to be used to supplement a TS canbe in the form of powder, suspension, strips or blocks or other forms asnecessary to perform its desired function.

As used herein, organoid means a structure that may be composed ofnatural, artificial, or a combination of natural and artificialelements, that wholly or in part, replaces the function of a naturalorgan. An example would be an artificial pancreas consisting of anetwork of capillaries surrounded by cells transfected with anexpression vector containing the gene for insulin. Such an organoidwould function to release insulin into the bloodstream of a patient withType I Diabetes.

Preparation of Supplemented TS

As a first step when practicing any of the embodiments of the inventiondisclosed herein, the supplement and TS must be selected. The supplementand TS may be prepared by methods known to those of skill in the art,may be purchased from a supplier thereof, or may be prepared accordingto the methods of this application. In a preferred embodiment, growthfactor, drug-or DBM-supplemented FG is prepared.

In any of the embodiments of the present invention the supplement may beadded to the fibrinogen, the thrombin, the calcium and/or the watercomponent(s) before they are mixed to form the TS. Alternatively, thesupplement(s) can be added to the components as they are being mixed toform the TS.

In embodiments of the present invention, the calcium and/or thrombin maybe supplied endogenously from body fluids as, for example, those in awound.

Preparation of TSs

In certain embodiments of this invention such as, but not limited to,vascular prostheses, and in bone and cartilage augmentation, TS whichallows cells to migrate into and/or through it may preferably be used.

Any TS, such as commercially available FG, may be used in someembodiments of this invention. For example, FGs which are well known tothose of skill in the art (see, e.g., U.S. Pat. Nos. 4,627,879;4,377,572; and 4,298,598, all herein incorporated by reference) may bepurchased from a supplier or manufacturer thereof, such as IMMUNO AG(Vienna, Austria) and BEHRINGWERKE AG (Germany). For these uses, such aslocalized drug delivery, the particular composition of the selected TSis not critical as long as it functions as desired. Commerciallyavailable FGs may be supplemented with growth factors, antibioticsand/or other drugs for use in the embodiments of this inventionincluding, but not limited to: in vitro cellular proliferation and/ordifferentiation; drug delivery; growth factor delivery, etc.

For the experiments exemplified herein, FG was prepared fromcryoprecipitate from fresh frozen plasma. The components of the FG thatwere used included: fibrinogen concentrate; thrombin; and calcium ions.

In a preferred embodiment of this invention, the total proteinconcentration in the prepared FG is from about 0.01 to 500 mg/ml of FG.In a more preferred embodiment, the total protein concentration in theprepared FG is from about 1 to 120 mg/ml FG. In the most preferredembodiment, the total protein concentration in the prepared FG is fromabout 4 to 30 mg/ml FG.

In a preferred embodiment of this invention, the fibrinogenconcentration used to prepare the FG is from about 0.009 to 450 mg/ml ofsolution. In a more preferred embodiment, the fibrinogen concentrationin this preparatory solution is from about 0.9 to 110 mg/ml. In the mostpreferred embodiment, the fibrinogen concentration in this preparatorysolution is from about 3 to 30 mg/ml.

In a preferred embodiment, the thrombin concentration used to preparethe FG is 0.01–350 U/ml. In a more preferred embodiment, the thrombinconcentration is 1–175 U/ml. In the most preferred embodiment, thethrombin concentration is 2–4 U/ml.

It is important that the calcium ion concentration be sufficient toallow for activation of the thrombin. In a preferred embodiment, the USPcalcium chloride concentration is 0–100 mM. In a more preferredembodiment, the USP calcium chloride concentration is 1–40 mM. In themost preferred embodiment, the USP calcium chloride concentration is 2–4mM. In some embodiments of this invention, the calcium may be suppliedby the tissue or body fluids as, for example, in the wound dressingembodiment.

In preparing the TS, sterile water for injection should be used.

Although the concentration(s) of growth factor(s), drugs and othercompounds will vary depending on the desired objective, theconcentrations must be great enough to allow them to be effective toaccomplish their stated purpose. In a preferred embodiment of thisinvention, the growth factor concentration is from about 1 ng/ml to 1mg/ml of FG. In a more preferred embodiment, the growth factorconcentration is from about 1 μg/ml to 100 μg/ml of FG. In the mostpreferred embodiment, the growth factor concentration is from about 5μg/ml to 20 μg/ml of FG. In a preferred embodiment of this invention theTET or CIP concentration is from 0.01 to 300 mg/ml FG. In a morepreferred embodiment of this invention the TET or CIP concentration is0.01–200 mg/ml. In the most preferred embodiment of this invention theTET or CIP concentration is 1–150 mg/ml. The amount of the supplementsto be added can be empirically determined by one of skill in the art bytesting various concentrations and selecting that which is effective forthe intended purpose and the site of application.

Preparation of Growth Factors

The growth factor(s), or mixture thereof, may be prepared by any methodknown to those of skill in the art or may be purchased commercially. Anygrowth factor may be selected including, but not limited to, forexample, growth factors that stimulate the proliferation and/orattraction of certain cell types, such as endothelial cells,fibroblasts, epithelial cells, smooth muscle cells, hepatocytes, andkeratinocytes, and/or growth factors which inhibit the growth of thesame cell types and smooth muscle cells. Such selection may be dependentupon the particular tissue site for which the growth factor-supplementedTS will be applied and/or the type of effect desired. For example, anEGF-supplemented TS may be preferred for application to wounds in theeye and for treating gastric ulcers while an osteogenin-supplemented TSmay be preferred for application to bone fractures and bone breaks inorder to promote healing thereof.

In another preferred embodiment HBGF-1β was prepared and added to FG.HBGF-1β, or HBGF-1α, or any other active form of HBGF-1,can be purifiedfrom natural sources, from genetically engineered cells that expressHBGF-1 or a derivative thereof, or by any method known to those of skillin the art.

HBGF-1β has been prepared using recombinant DNA methodology (Jaye etal., U.S. Pat. No. 4,868,113; Jaye et al., J. Biol. Chem.262:16612–16617 (1987)). Briefly, DNA encoding HBGF-1β was cloned into aprokaryotic expression vector, a pUC9 derivative, and expressedintracellularly in E. coli. The expressed peptide was then released fromthe cells by pressure, using a cell disrupter operated on highcompression-decompression cycles. After disruption, cell debris wasremoved by filtration and HBGF-1β was purified from the supernatantusing standard methods of protein purification including affinitychromatography on heparin Sepharose™ followed by ion-exchangechromatography on CM-Sepharose™.

In addition to HBGF-1, described above, other growth factors that may beadded to the FG include, but are not limited to, HBGF-2, IGF-1, EGF,TGF-β, TGF-α, any platelet-derived growth factor or extract, BMPs, andmixtures of any growth factors. For example, platelet-derived extracts,which serve as rich sources of growth factors, may be added to the TS inaddition to or in place of other growth factors, such as HBGF-1.

In a preferred embodiment, a platelet-derived extract, prepared by anymethod known to those of skill in the art, is added to a TS. Such anextract has been prepared from plasma derived platelets for use with FG.

Platelet-Derived Wound Healing Factor (PDWHF) may be prepared and addedto FG (Knighton et al., Ann. Surg. 204:322–330 (1986)). Briefly, toprepare PDWHF, blood is drawn into anticoagulant solution andplatelet-rich plasma is prepared by refrigerated centrifugation. Theplatelets are isolated and stimulated with thrombin, which releases thecontents of the alpha granule contents. The platelets are removed and aneffective concentration of the remaining extract is added to a TS.

Additional Components of Growth Factor-Supplemented TS

Since they are essentially plasma fractions, the TSs contemplated foruse with growth factors contain numerous components, some of which mayinterfere with the biological activity of the selected growth factor(s).For example, thrombin, which is an essential component of FG, can act asa proteolytic enzyme and specifically cleave HBGF-1β. Therefore, it maybe necessary to include additional compounds, such as protease or otherinhibitors, that protect the selected growth factor(s) from the actionof other components in the TS which interfere with or destroy thebiological activity of the growth factor(s).

Selection of the particular inhibiting compound(s) may be empiricallydetermined by using methods, discussed below, that assess the biologicalactivity of the growth factor(s) in the TS. Methods to assess biologicalactivity are known to those of skill in the art.

In addition, in order for certain growth factors to exhibit theirbiological activities, it may be necessary to include compounds thatpotentiate or mediate the desired activity. For example, heparinpotentiates the biological activity of HBGF-1 in vivo (see, e.g.,Burgess et al., Annu. Rev. Biochem. 58:575–606 (1989)).

The supplemented TS of the present invention may contain compounds suchas drugs, other chemicals, and proteins. These may include, but are notlimited to: antibiotics such as TET, ciprofloxacin, amoxicillin, ormetronidazole, anticoagulants, such as activated protein C, heparin,prostracyclin (PGI₂), prostaglandins, leukotrienes, antithrombin III,ADPase, and plasminogen activator; steroids, such as dexamethasone,inhibitors of prostacyclin, prostaglandins, leukotrienes and/or kininsto inhibit inflammation; cardiovascular drugs, such as calcium channelblockers; chemoattractants; local anesthetics such as bupivacaine; andantiproliferative/antitumor drugs such as 5-fluorouracil (5-FU), taxoland/or taxotere. These supplemental compounds may also includepolyclonal, monoclonal or chimeric antibodies, or functional derivativesor fragments thereof. They may be antibodies which, for example, inhibitsmooth muscle proliferation, such as antibodies to PDGF, and/or TGF-β,or the proliferation of other undesirable cell types within and aboutthe area treated with the TS. These antibodies can also be useful insituations where anti-cancer, anti-platelet or anti-inflammatoryactivity is needed. In general, any antibody whose efficacy would beimproved by site-directed delivery may benefit from being used with thisTS delivery system.

Assays for Assessing the Wound Healing Properties of a GrowthFactor-Supplemented TS

In order to ascertain whether a particular growth factor-supplemented TSpromotes wound healing and to select optimal concentrations of thegrowth factor(s) to do the same, the composition may be tested by anymeans known to those of skill in the art (see, e.g., Tsuboi et al., J.Exp. Med. 172:245–251 (1990); Ksander et al., J. Am. Acad. Dermatol.22:781–791 (1990); and Greenhalgh et al., Am. J. Path. 136:1235 (1990)).Any method including both in vivo and in vitro assays, by which theactivity of the selected growth factor(s) in the TS composition can beassessed may be used. For example, the activity of HBGF-1β has beenassessed using two independent in vitro assays. In the first, theproliferation of endothelial cells that had been suspended in a shallowfluid layer covering a plastic surface which had been impregnated withgrowth factor-supplemented FG was measured. In the second, theincorporation of ³H-thymidine in cultured fibroblasts in the presence ofHBGF-1 was measured.

In an in vivo assay, FG that had been supplemented with HBGF-1β has beentested for its ability to promote healing in vivo using mice as a modelsystem. In this method identical punch biopsies were made in the dorsalregion of the mice, which were then separated into test, treated controland untreated control groups. The wounds in the mice in the test groupwere treated with the growth factor-supplemented TS. The wounds in themice in the treated control group were treated with unsupplemented TS.The wounds in the untreated group were not treated with TS. After a timesufficient for detectable wound healing to proceed, generally a week toten days, the mice were sacrificed and the wound tissue wasmicroscopically examined to histologically assess the extent of woundrepair in each group.

The ability of the growth factor-supplemented TS to induce cellproliferation and to recruit cells may also be assessed by in vitromethods known to those of skill in the art. For example, the in vitroassays described above for measuring the biological activity of growthfactors and described in detail in the Examples, may be used to test theactivity of the growth factor in the TS composition. In addition, theeffects of adding inhibiting and/or potentiating compounds can also beassessed.

Generally, the necessity for adding inhibiting and/or potentiatingcompounds can be empirically determined. For example, in the experimentsdescribed below, the HBGF-1β in HBGF-1-supplemented FG was specificallycleaved in a stochastic manner, suggesting that a component of the FGpreparation, most likely thrombin, was responsible. Heparin, which isknown to bind to HBGF-1 and protect it from certain proteolyticactivities, was added to the HBGF-1-supplemented FG. The addition ofrelatively low concentrations of heparin protected HBGF-1β from cleavagethat would destroy its biological activity in the FG. Therefore, TScompositions that include HBGF-1 may include heparin or some othersubstance that inhibits the cleavage of HBGF-1 by thrombin or otherproteolytic components of the FG.

Similarly, the ability of a selected inhibitor to protect a growthfactor from degradation by TS components may be assessed by any methodknown to those of skill in the art. For example, heparin has been testedfor its ability to inhibit cleavage of HBGF-1 by thrombin, which is anessential component of FG. To do so, mixtures of various concentrationsof heparin and HBGF-1-supplemented FG have been prepared, and incubatedfor various times. The biological activity of HBGF-1 in the mixture hasbeen tested and the integrity of the HBGF-1 has been ascertained usingwestern blots of SDS gels. Relatively low concentrations, about a 1:1molar ratio of heparin:HBGF-1, are sufficient to protect HBGF-1 fromdegradation in FG.

It can also be empirically determined whether a particular compound canbe used to potentiate, mediate or enhance the biological activity of agrowth factor(s) in TS.

Topical or Internal Application of the Growth Factor-Supplemented TS toan Internal or External Wound

Prior to clinical use, the growth factor and TS, or the growthfactor-supplemented TS is pasteurized or otherwise treated to inactivateany pathogenic contaminants therein, such as viruses. Methods forinactivating contaminants are well-known to those of skill in the artand include, but are not limited to, solvent-detergent treatment andheat treatment (see, e.g., Tabor et al., Thrombosis Res. 22:233–238(1981) and Piszkiewicz et al., Transfusion 28:198–199 (1988)).

The supplemented TS is applied directly to the wound, other tissue orother desired location. Typically for external wounds it can be applieddirectly by any means, including spraying on top of the wound. It canalso be applied internally, such as during a surgical procedure. When itis applied internally, such as to bones, the clot gradually dissolvesover time.

Self-Contained Applications of the Supplemented or Unsupplemented TS forInternal or External Wounds

The TSs may be formulated as a self-contained wound dressing, or fibrinsealant bandage, which contains the necessary thrombin and fibrinogencomponents of the FG. The self-contained dressing or bandage iseasy-to-use, requiring no advanced technical knowledge or skill tooperate. It can even be self-administered as an emergency first aidmeasure to preserve life until medical assistance becomes available.

The self-contained TS wound dressing or fibrin sealant bandage is anadvancement over the current technology in that the field-readypreparation can be stored for long periods, and be used to provide rapidTS treatment of a hemorrhaging wound without the time delay associatedwith solubilization and mixing of the components. These characteristicsmake it ideal for use in field applications, such as in trauma packs forsoldiers, rescue workers, ambulance/paramedic teams, firemen, and inearly trauma and first aid treatment by emergency room personnel inhospitals and clinics, particularly in disaster situations. A smallversion may also have utility in first aid kits for use by the generalpublic or by medical practitioners.

The self-contained TS wound dressing or fibrin sealant bandage comprisesa tissue sealing composition comprising a tissue sealant or fibrincomplex of the type previously described. For example, the compositionmay be comprised of purified fibrinogen, thrombin and calcium chloridewith sufficient Factor XIII to produce a fibrin clot. In one embodimentthe fibrinogen and Factor XIII components are supplied in the form oftopical fibrinogen complex (TFC).

When used on human patients, the components are most preferablypathogen-inactivated, purified components derived from human sources. Inparticular, the components of the present invention, including additivesthereto, are treated with a detergent/solvent, and/or otherwise treated,e.g., by pasteurization or ultrafiltration to inactivate any pathogeniccontaminants therein, such as viruses. Methods for inactivatingcontaminants are well-known to those of skill in the art and include,but are not limited to, solvent-detergent treatment and heat treatment.Solvent-detergent treatment is particularly advantageous in that theproteinaceous components are not exposed to irreversibleheat-denaturation.

The calcium and/or Factor XIII components may be contained in either thethrombin and/or the fibrinogen component(s), and/or absorbed from thepatient's endogenous calcium present in the fluids escaping from thewound. Thrombin may also be supplied endogenously. Either or both of thethrombin or fibrinogen components can be, but does not have to be,supplemented in each of the following embodiments with one or moregrowth factors, drugs, inhibiting compounds (to inhibit the activitiesof the sealant that may interfere with any of the biological activitiesof the growth factor or drug), and potentiating compounds (topotentiate, mediate or enhance any of the biological activities of thegrowth factor or drug), compounds which inhibit the breakdown of thefibrin clot, or dyes.

The growth factor may include, e.g., fibroblast growth factor-1,fibroblast growth factor-2 and fibroblast growth factor-4;platelet-derived growth factor; insulin-binding growth factor-1;insulin-binding growth factor-2; epidermal growth factor; transforminggrowth factor-α; transforming growth factor-β; cartilage-inducingfactors-A and -B; osteoid-inducing factor; osteogenin and other bonegrowth factors; collagen growth factor; heparin-binding growth factor-1;heparin-binding growth factor-2; and/or their biologically activederivatives.

The drug may be an analgesic, antiseptic, antibiotic or other drug(s),such as antiproliferative drugs which can inhibit infection, promotewound healing and/or inhibit scar formation. More than one drug may beadded to the composition, to be released simultaneously, or the drug maybe released in predetermined time-release manner. Such drugs mayinclude, for example, taxol, tetracycline free base, tetracyclinehydrochloride, ciprofloxacin hydrochloride or 5-fluorouracil. Theaddition of taxol to the fibrin sealant complex may be particularlyadvantageous. Further, the drug may be a vasoconstrictor, e.g.,epinephrine; or the drug may be added to stabilize the tissue sealant orfibrin clot, e.g., aprotinin. The supplement(s) is at a concentration inthe TS such that it will be effective for its intended purpose, e.g., anantibiotic will inhibit the growth of microbes, an analgesic willrelieve pain, etc.

Dyes, markers or tracers may be added, for example, to indicate theextent to which the fibrin clot may have entered the wound, or tomeasure the subsequent resorption of the fibrin clot, or the dye may bereleased from the tissue sealant in a predetermined, time-release mannerfor diagnostic purposes. The dyes, markers or tracers must bephysiologically compatible, and may be selected from colored dyes,including water soluble dyes, such as toluidine blue, and radioactive orfluorescent markers or tracers which are known in the art. The dyes,markers or tracers may also be compounds which may be chemically coupledto one or more components of the tissue sealant. In addition, the markermay be selected from among proteinaceous materials which are known inthe art, which upon exposure to proteolytic degradation, such as wouldoccur upon exposure to proteases escaping from wounded tissue, changecolor or develop a color, the intensity of which can be quantified.

Moreover, when the TS is used to replace or repair wounded or damagedbone or ossified tissue, the composition may also be supplemented witheffective amounts of demineralized bone matrix and/or bone morphogenicproteins, and/or their biologically compatible derivatives.

The concentration of the fibrinogen and/or thrombin components of theself-contained TS wound dressing or fibrin sealant bandage may have asignificant effect on the density and clotting speed of the final fibrinmatrix. This principle may be used to satisfy specific uses of theself-contained TS wound dressing or fibrin sealant bandage inspecialized situations. For example, the treatment of an arterial woundmay require the fibrin clot to set very rapidly and with sufficientintegrity to withstand pressurized blood flow. On the other hand, whenfilling deep crevices in a wound, treatment may require the componentsto fill the wound completely before the fibrin clot sets.

The Gel Pack Embodiments

In the gel pack embodiment of the self-contained dressing, the thrombinand fibrinogen components are individually contained in independentquick-evaporating gel layers (e.g., methylcellulose/alcohol/water),wherein the two gel layers are separated from each other by animpermeable membrane, and the pair are covered with an outer,protective, second impermeable membrane. The bandage may be coated onthe surface that is in contact with the gel in order to insure that thegel pad remains in place during use. (See FIG. 42).

In use, the membrane separating the two gel layers is removed, allowingthe two components to mix. The outer membrane is then removed and thebandage is applied to the wound site. The action of the thrombin andother components of the fibrinogen preparation cause the conversion ofthe fibrinogen to fibrin, in the manner previously disclosed for otherFS applications. This results in a natural inhibition of blood and fluidloss from the wound, and establishes a natural barrier to infection.

In a similar gel pack embodiment, both the thrombin component, and theplastic film separating the thrombin gel and the fibrinogen gel, may beomitted. In operation, the outer impervious plastic film is removed andthe bandage applied, as previously described, directly to the woundsite. The thrombin and calcium naturally present at the wound site theninduce the conversion of fibrinogen to fibrin and inhibit blood andfluid loss from the wound as above.

This alternative embodiment of the gel pack has the advantage of beingsimpler, cheaper, and easier to produce. However, there may becircumstances in which a patient's wounds have insufficient thrombin toeffectively transform the fibrinogen gel into a fibrin tissue sealant.In those cases, the thrombin component must be exogenously supplied, asin the earlier-described gel pack embodiment of the invention.

The Fibrin Sealant Bandage Embodiments

A fibrin sealant bandage embodiment is formulated for applying a tissuesealing composition to wounded tissue in a patient, wherein the bandagecomprises, in order: (1) an occlusive backing; (2) aphysiologically-acceptable adhesive layer on the wound-facing surface ofthe backing; and (3) a layer of dry materials comprising an effectiveamount, in combination, of (a) dry, virally-inactivated, purifiedfibrinogen complex, (b) dry, virally-inactivated, purified thrombin, andas necessary (c) effective amounts of calcium and/or Factor XIII toproduce a tissue-sealing fibrin clot upon hydration, wherein the layerof dry materials is affixed to the wound-facing surface of the adhesivelayer. In one embodiment, the occlusive backing and thephysiologically-acceptable adhesive layer are one and the same, if thebacking layer is sufficiently adhesive to effectively bind the layer ofdry materials.

In another embodiment, are movable, waterproof, protective film isplaced over the layer of dry materials and the exposed adhesive surfaceof the bandage for long-term stable storage. In operation thewaterproof, protective film is removed prior to the application of thebandage over the wounded tissue.

The tissue sealant component of the bandage in one embodiment isactivated at the time the bandage is applied to the wounded tissue toform a tissue sealing fibrin clot by the patient's endogenous fluidsescaping from the hemorrhaging wound. Preferably, the tissue sealant ishydrated and fluid loss from the wound will be significantly diminishedwithin minutes of application of the bandage to the wounded tissue.Although the speed with which the fibrin clot forms and sets may be tosome degree dictated by the application, e.g., rapid setting forarterial wounds and hemorrhaging tissue damage, slower setting fortreatment of wounds to bony tissue, preferably the fibrin clot will formwithin twenty minutes after application. More preferably, this effectwill be evident within ten minutes after application of the bandage.Most preferably, the fibrin clot will form within two to five minutesafter application. In the embodiment comprising the most rapidly formingfibrin clot, the tissue seal will be substantially formed within 1–2minutes, more preferably within 1 minute, and most preferably within 30seconds after application.

It may be necessary to use pressure in applying the fibrin sealantbandage until the tissue sealing fibrin clot has formed over the woundsite.

In the alternative, in situations where fluid loss from the wound isinsufficient to provide adequate hydration of the dry tissue sealantmaterials, or where time is of the essence, as in a life-threateningsituation, the tissue sealant components are hydrated by a suitable,physiologically-acceptable liquid prior to application of the bandage tothe wounded tissue.

To construct the bandage, the dry materials may be obtained, forexample, by lyophilization or freeze-drying, or suitable,commercially-available materials may be utilized. Anhydrous CaCl₂ mayalso be added to the dry TS components to accelerate the speed of fibrinformation upon hydration of the fibrin sealant bandage. The binding ofthe dry materials to the adhesive or backing layer may be enhanced byadding a binder, preferably a water soluble binder, to the drycomponents.

The backing of the fibrin sealant bandage may be of conventional,non-resorbable materials, e.g., a silicone patch or plastic material; orit may be of biocompatible, resorbable materials. The backing materialmay act as more than a delivery device. Its preferred composition isdetermined by the desired application of the fibrin sealant bandage. Forexample, a non-resorbable backing is appropriate for many external uses,where it provides strength and protection for the fibrin clot. In analternative embodiment, the non-resorbable backing is reinforced, e.g.,with fibers, to provide extra strength and durability for the protectivecovering over the fibrin clot.

Subsequent removal of the clot with the backing is acceptable in manysituations, such as when the fibrin sealant bandage is used as a firstaid measure until medical assistance becomes available. In such asituation, the clot will have served its purpose to prevent lifethreatening loss of fluid, and it will be desirable to remove the clotwithout causing additional tissue damage to permit proper treatment orsurgical repair of the wound.

In the alternative, the non-resorbable backing may be used to providestrength to the tissue sealing fibrin clot during its formation, e.g.,when the hemorrhaging fluids are escaping under pressure, as in anarterial wound. Yet, if such a wound is internal, it is advantageous toremove the backing from the fibrin clot without disturbing the tissueseal. Therefore, a fibrin sealant bandage is provided in which theadhesive layer is of a material having a lower shear strength than thatof the fibrin clot, permitting removal of the backing without damage tothe fibrin clot or the tissue surrounding the wound.

By comparison, certain internal applications mandate the use of aresorbable backing to eliminate the need for subsequent removal of thedressing. A resorbable material is one which is broken downspontaneously or by the body into components which are consumed oreliminated in such a manner as to not significantly interfere withhealing and/or tissue regeneration or function, and without causing anyother metabolic disturbance. Homeostasis is preserved. Materialssuitable for preparing the biodegradable backing include proteinaceoussubstances, e.g., fibrin, collagen, keratin and gelatin, or carbohydratederived substances, e.g., chitin, chitosan, carboxymethylcellulose orcellulose, and/or their biologically compatible derivatives.

The adhesive layer, if separate from the occlusive backing layer, isselected on the basis of the intended application of the fibrin sealantbandage, and may comprise conventional adhesive materials. Antisepticmay be added to the adhesive layer.

If the tissue sealing fibrin clot is to be removed from the wound withthe occlusive backing, such as prior to surgery, the adhesive must besufficient to affix the dry material layer to the occlusive backing, andto maintain an adhesive capability after hydration which is greater thanthe sheer strength of fibrin.

If the tissue sealing fibrin clot is to remain in position over thewound, but the occlusive backing must be removed after application, theadhesive must be sufficiently sticky to affix the dry material layer tothe occlusive backing, but yet have an adhesive capability afterhydration which is less than the sheer strength of the fibrin clot. Inthe alternative, the adhesive layer may be of a material which becomessolubilized or less sticky during hydration of the dry materials,permitting removal of the backing from the fibrin clot. In thealterative for such purposes, the dry material layer may be affixeddirectly to the occlusive bandage.

In another embodiment, the adhesive layer comprises two differentadhesives to permit removal after hydration of the occlusive layerwithout disturbing the tissue sealing fibrin clot. Typically, in such asituation the dry, tissue-sealant component materials are affixed to aspecific region of the backing, the “inner region,” e.g., the center,with an unencumbered area of adhesive extending beyond the area of drymaterial, the “outer region.”

The outer region of adhesive is affixed directly to the skin or tissuesurrounding or adjacent to the wound in such a way that the dry materialregion of the bandage forms a fibrin clot directly over the wound. Theadhesive layer on the region of backing which is not covered by the drymaterial layer of the bandage is sufficient to affix the fibrin sealantbandage to the tissue surrounding the wound until its physical removal.The adhesive on the outer region must be sufficient to hold the bandagein place, even if fluids are hemorrhaging from the wound under pressure,e.g., an arterial wound.

The inner region of adhesive is sufficiently sticky to affix the drymaterial layer to the occlusive backing, but yet have an adhesivecapability after hydration which is less than the sheer strength of thefibrin clot. In the alternative, the inner region of adhesive is of amaterial which becomes solubilized or less sticky during hydration ofthe dry materials, permitting removal of the backing from the fibrinclot. In the alterative for such purposes, the dry material layer may beaffixed in the inner region directly to the occlusive bandage, with anadhesive layer added only to the outer layer.

Thus, in the two adhesive embodiment, the backing of the fibrin sealantbandage remains in place affixed to the tissue surrounding the wounduntil the bandage is physically removed. But upon removal, the backingseparates from the tissue sealing fibrin clot without disturbing thetissue seal.

The Dual-Encapsulated Embodiments of the Fibrin Sealant Bandage

In yet another embodiment of the fibrin sealant bandage, an independenthydrating layer comprising an effective amount of carbonated water orphysiologically-acceptable buffered hydrating agent, such as PBS, orcomparable gel, is contained within a rupturable, liquid-impermeablecontainer. The rupturable, liquid-impermeable container encapsulatingthe hydrating layer is affixed directly to the above-described occlusivebandage layer or to the above-described adhesive layer adjacent to theocclusive bandage. Affixed to the exposed side (the side which is notattached to the backing or adhesive layer) of the rupturable,liquid-impermeable container encapsulating the hydrating layer is a drylayer of finely-ground, powdered fibrin components, as described above.The layer of dry components includes powdered fibrinogen or fibrinogencomplex, thrombin, and as necessary sufficient calcium and/or FactorXIII to, upon hydration, form a fibrin clot.

The dual layers (the dry layer and the hydrating layer) are togethercovered on all surfaces not in contact with the occlusive backing oradhesive material affixing the layers to the occlusive backing, with anouter, protective, second impermeable membrane. Thus, in this dual-layerembodiment, the contents are entirely encapsulated within an impermeablecontainer, wherein one side is the occlusive backing material and theother side and all edges are formed by the outer, protective, secondimpermeable membrane.

In operation the inner liquid-impermeable container encapsulating thehydrating layer is physically ruptured to release the hydrating materialcontained therein into the dry fibrin component layer, resulting in afully-hydrated tissue sealing fibrin clot to inhibit blood and fluidloss from the wound, and to provide a natural barrier to infection. Theouter, second impermeable membrane retains the released hydratingmaterial in contact with the dry components until a malleable fibrincomplex forms, at which time the outer membrane is physically removedand the bandage placed over the wound to form a tissue sealant.

In the alternative, the outer membrane may be physically removed, andthe dual layers forcefully applied to the wound area in a manner whichruptures the inner liquid-impermeable container and releases thehydrating agent into the dry fibrin components so that the tissuesealing fibrin clot is formed directly on the wounded tissue.

As in other embodiments of the fibrin sealant bandage, the selectedadhesives and backing materials may be determined by the intendedapplication of the bandage. The backing may be removable or resorbable,and the adhesive may have the intended purpose upon removal of thebandage of removing the tissue sealant from the wound, or of leaving thetissue sealing fibrin clot undisturbed. The adhesive may be a separatelybound layer, or the backing may itself act as an adhesive to affix thedry fibrin components.

The thrombin, calcium and Factor XIII components which are necessary toform the fibrin complex may be affixed as dry material(s) in the drymaterial layer, or they may be included in liquid or gel form in thehydrating layer. Moreover, they may be divided between the two layers,so long as all of the necessary fibrin-forming components are present,and the dry layer remains non-hydrated until the bandage is used. Inaddition, additives, such as the previously disclosed growth factors,antibiotics, antiseptics, antiproliferative drugs, etc. may also beincluded in this embodiment of the fibrin sealant bandage.

If the hydrating layer contains a liquid supersaturated with gas, thedry material layer will be hydrated as an expandable, foaming, fibrintissue sealant. In the alternative, the dry material layer may besupplemented with materials which produce gas, and hence foaming, uponcontact with the hydrating agent.

If the hydrating layer is in the form of a gel, such as aquick-evaporating gel layers (e.g., methylcellulose/alcohol/water), therupture of the surrounding impermeable barrier permits the dry materialfibrin components to directly contact the hydrating layer as disclosedabove to produce the tissue sealing fibrin clot. The gel layer, in themanner described for a liquid hydrating layer, may comprise any one, orall, of the thrombin, calcium or Factor XIII elements of the fibrincomplex, and/or any one of the above-disclosed additives.

In an alternate dual layer embodiment, the tissue sealant is deliveredas a wound sealing dressing, which need not be affixed to a backing. Thecomponents are organized essentially as a capsule within a capsule,wherein the term capsule is used to define a broad concept, rather thana material. The above-described encapsulated hydrating layer is itselfcontained within a second encapsulating unit, which contains both thedry fibrin component materials and the encapsulated hydrating layer.

In operation, the inner, liquid-impermeable container encapsulating thehydrating layer is physically ruptured to release the hydrating materialcontained therein into the dry fibrin component layer, both of whichremain completely contained within the outer, second encapsulating unit.The integrity of the outer, second encapsulating unit is not broken whenthe inner container encapsulating the hydrating layer is physicallyruptured.

The mixing of the hydrating layer with the dry fibrin components withinthe outer encapsulating unit results in a fully-hydrated tissue sealingfibrin clot, which is then released or expelled onto wounded tissue toform a tissue seal. To release the fibrin mass, the outer encapsulatingunit is physically cut or torn, either randomly or at a specificlocation on the surface, e.g., to form a pour spout to direct the flowof the malleable fibrin mass onto the wound site.

If the hydrating layer is a agent supersaturated with gas, the mixing ofthe hydrating agent with the dry fibrin components results in anexpandable foaming mixture, which is then applied to the wounded tissue.The foaming may, in the alternative, be achieved by hydration of the drycomponent layer.

The Self-Foaming Fibrin Sealant Embodiments

A self-foaming fibrin sealant dressing embodiment for treating woundedtissue in a patient is formulated as an expandable foam comprising afibrin-forming effective amount, in combination, of (1)virally-inactivated, purified fibrinogen, (2) virally-inactivated,purified thrombin, and as necessary (3) calcium and/or Factor XIII;wherein said composition does not significantly inhibit full-thicknessskin wound healing. The previously described TS components are stored ina canister or tank with a pressurized propellant, so that the componentsare delivered to the wound site as an expandable foam, which will withinminutes form a fibrin seal.

Acceptable formulations of the expandable foam embodiment provide thehydrated components of a fibrin clot, which in operation expand up totwenty-fold. The extent of expansion of the tissue sealing fibrin clot,however, is determined by its intended application.

For example, use of the expandable foam fibrin sealant dressing withinthe abdomen provides a fibrin tissue sealant to significantly diminishor prevent blood or fluid loss from injured internal tissues organs orblood vessels, while also providing a barrier to infection. However, atthe same time the expansion of the foam must be controlled to preventharmful pressure on undamaged tissue, organs or blood vessels. Such asituation may warrant the use of an expandable foam dressing in whichthe expansion is limited to only 1- or 2-fold, and not more than 5–10fold.

By comparison, use of the expandable foam fibrin sealant dressing tofill gaps within bone, may warrant the use of material which expands ata much greater rate to produce a tight and firm seal over the woundedarea. Arterial wounds may also respond well to a highly pressurized foamtissue sealant dressing. The extent of the expansion of such materialmay be in the range of above 20-fold, although preferably 10–20 fold, ormore preferably 5–10 fold. An expansion of less than 5-fold, including1- to 2-fold may also be applicable to repair of blood vessels orinjured bone, for example in small areas, such as the inner ear.

Like the expansion rate, the set-up time for the formation of the fibrinseal using the expandable foam fibrin dressing is also related to itsintended application. In certain situations loss of life may beimminent, such as in a patient who has suffered arterial wounds ordamaged heart tissue. In such a situation the fibrin dressing mustexpand very rapidly and form the fibrin tissue seal as quickly aspossible, necessarily before exsanguination. Preferably the seal willset-up and significantly diminish the patient's fluid loss within 2minutes or less, more preferably in 1–2 minutes, and most preferably inless than 1 minute.

On the other hand, not all wounds are immediately life threatening. Forexample, the strength of the tissue sealant repair of bony tissue ismore important than a rapid set-up time. In such situations, thecomposition of the tissue sealing fibrin clot may be modified to permitgreater cross-linking or thickening of the fibrin fibrils, or to permitdelivery of a more dilute composition which will continue to expand fora longer period of time. Such formulations may either permit or requirea slightly longer time to set-up the tissue sealing fibrin clot.Although a set-up time of under 1 minute is appropriate for suchapplications, set-up times of 1–2 minutes, or up to 5 minutes would beacceptable. In circumstances recognizable to one of ordinary skill inthe art, a long set-up time of 5–10 minutes, or even up to twentyminutes, may be acceptable in non-life threatening situations.

The delivery devices, e.g., canister, tank, etc., may be developedespecially for the present application, or they may be commerciallyavailable. The canister may comprise either a single or multiplereservoirs. Separate reservoirs, although more expensive, willadvantageously permit the hydrated components to remain separated andstable until they are mixed upon application.

The propellant must be physiologically acceptable, suitable forpharmacological applications, and may include conventionally recognizedpropellants, for example, CO₂, N₂, air or inert gas, such as freon,under pressure. In the alternative, the dry fibrin components may besupplemented with material(s) which produce gas, and hence foaming, uponcontact with the hydrating agent.

Since delivery pressure of the expandable foam fibrin dressing from thedelivery device, when combined with the composition of the fibrin clotitself and its set-up time, determines the extent of expansion of thedressing, the delivery pressure is determined by the nature of the woundbeing treated. As described above, certain wounds require immediateformation of the tissue sealing fibrin clot to prevent loss of life,while others wounds require slow delivery or time to form extensivecross-links to strengthen the tissue sealing composition. Therefore,delivery pressure may ideally be situation specific.

Pressure of 1 atmosphere, or less (14.7 lbs/inch²) will provide a lowlevel of expansion and a slower rate of delivery. However, certain lifethreatening situations may warrant a delivery pressure of 1–5atmospheres, or more. In most cases, the delivery pressure chosencorresponds to that of commercially available canister devices. As anaddition factor, the delivery pressure may be important to keep thetissue sealant material from clogging delivery lines or devices.

Combined Embodiments of the Self-Contained Wound Dressing and FibrinSealant Bandage

Finally, certain traumatic injuries will be best treated by combiningseveral embodiments of the self-contained fibrin sealant dressing. Forexample, in serious car accidents or injuries caused byantipersonnel-mines or explosives, the wounds may be not onlylife-threatening but extensive, involving large, jagged openings intissue or bone with significant internal damage, often with accompanyingserious burns. Such wounds may present numerous severed arteries andblood vessels in addition to extensive areas of wounded tissue. In suchwounds, it may be advantageous to first liberally apply a rapidlysetting expandable fibrin foam dressing to quickly control hemorrhaging,and then to wrap the entire area in an embodiment of the fibrin sealantbandage to support and protect the wounded area and seal slow fluid lossfrom, for example, burned tissue, until the victim can be transported toa medical facility, or until professional medical assistance canadministered. In most instances, additional formulations of the fibrinsealant dressing will then be applied by the trained personnel for thelong-term repair, treatment and protection of the injured tissue.

The following examples are included for illustrative purposes only andare not intended to limit the scope of the invention.

EXAMPLE 1 Preparation of HBGF-1 for Supplementation of FG

An 800 ml culture of recombinant E. coli containing a plasmid thatincluded DNA encoding HBGF-1β was prepared. After induction andculturing for 24 hours at 37° C., the cells were centrifuged and thesupernatant was discarded. The cell pellet was resuspended in 25 mls of20 mM phosphate buffer, containing 0.15 M NaCl, pH 7.3. The suspendedcells were disrupted with a cell disrupter and the cell debris wasseparated from the resulting solution by centrifugation at 5000 g for 20min.

The pellet was discarded and the supernatant containing the solubilizedHBGF-1β and other bacterial proteins was loaded onto a 2.6 cm diameterby 10 cm high column of Heparin-Sepharose™ (Pharmacia Fine Chemicals,Upsala, Sweden). The column was washed with 5 column volumes of 0.15 MNaCl in 20 mM phosphate buffer, pH 7.3, and then was eluted with a 0.15M NaCl in 20 mM phosphate buffer to 2.0 M NaCl gradient.

The eluate was monitored by UV absorption at 280 nm. Three peaks of UVabsorbing material eluted and were analyzed by SDS polyacrylamide gelelectrophoresis. Peak number three electrophoresed as a single band atabout 17,400 daltons and contained substantially pure HBGF-1β.

In order to further insure that the HBGF-1β was free of contaminatingbacterial proteins, peak number three, which contained the growth factoractivity, was dialyzed overnight against 20 mM histidine, 0.15 M NaCl,pH 7.5. Two mg of protein was loaded onto a 1 ml CM-Sepharose™(Pharmacia, Upsala, Sweden) ion exchange column. The column was washedwith 10 bed volumes (0.5 ml/min) of 20 mM histidine, 0.15 M NaCl, pH 7.5and eluted with a gradient of 0.15 M NaCl to 1.0 M NaCl in 20 mMhistidine, pH 7.5. The eluate was monitored by UV absorption at 280 nmand HBGF-1β was identified by SDS polyacrylamide gel electrophoresis.

This purified HBGF-1 was used to supplement FG in subsequent examples.

EXAMPLE 2 Stability of HBGF-1

It was necessary to add an ingredient to the FG that would inhibit orprevent the digestion of HBGF-1β by thrombin (Lobb, Biochem.27:2572–2578 (1988)), which is a component of FG. Heparin, which adsorbsto HBGF-1, was selected and tested to determine whether it could protectHBGF-1 from digestion by thrombin and any other proteolytic componentsof the FG. The stability of HBGF-1 in the presence of increasingconcentrations of heparin was assessed.

Solutions containing HBGF-1β (10 μg/ml), thrombin (250 U/ml), andincreasing concentrations of heparin (0, 0.5, 5, 10, 20, and 50 U/ml)were incubated at 37° C. Aliquots were periodically removed from theincubating solutions and were frozen and stored at −70° C. for furthertesting.

After the incubation was complete, the samples were thawed and separatedon 15% SDS polyacrylamide gels under reducing conditions according tothe method of Laemmli (Nature 227:680 (1970)). The gel was thenelectroblotted onto nitrocellulose and the band corresponding to HBGF-1was identified using an affinity-purified polyclonal rabbit antiserum toHBGF-1. The Western blots are shown in FIG. 1 on which the HBGF-1β bandat 17,400 mw can be seen. The results indicated that in the presence ofconcentrations of heparin as low as 5 U/ml, HBGF-1β was protected fromdigestion by thrombin. In addition, as described in Example 3, itsbiological activity was not altered.

EXAMPLE 3 The Biological Activity of HBGF-1β after Incubation in thePresence of Heparin and Thrombin

The biological activity of HBGF-1 in the incubation mixture thatcontained 5 U/ml of heparin, and was described in Example 2, wasmeasured using an ³H-thymidine incorporation assay with NIH 3T3 cells.

NIH 3T3 cells were introduced into 96 well plates and were incubated at37° C. under starvation conditions in Dulbecco's Modified Medium (DMEM;GIBCO, Grand Island, N.Y.) with 0.5% fetal bovine serum (BCS; GIBCO,Grand Island, N.Y.) until the cells reached 30 to 50% confluence. Twodays later, varying dilutions of HBGF-1 from the samples prepared inExample 2 were added to each well without changing the medium. Diluent(incubation buffer) was added in place of growth factor for the negativecontrols and DMEM with 10% BCS, which contains growth factors needed forgrowth, was added in place of the HBGF-1 sample for the positivecontrols.

After incubation at 37° C. for 18 hours, 0.25 μCi of ³H-thymidine,specific activity 6.7 μCi/mol, was added to each well and the incubationwas continued at 37° C. for an additional 4 hours. The plates wererinsed with phosphate-buffered saline (PBS) and fixed with 0.5 ml cold10% trichloracetic acid (TCA) for 15 min at 4° C. The TCA was removed,the plates were rinsed with PBS and the acid-precipitable material wassolubilized with 0.5 ml/well of 0.1 N sodium hydroxide for 1 hour atroom temperature. The samples were transferred to scintillation vialsand 10 ml of scintillation fluid (New England Nuclear, Aquasure™) wasadded per vial.

The results, which are shown in FIG. 2, demonstrated that HBGF-1, whichhad been incubated in the presence of thrombin and heparin, retained itsbiological activity. The observed concentration dependence of thymidineincorporation was independent of incubation time and was typical of thatexpected for the dependence of the proliferation of cells as a functionof growth factor concentration. Growth factors typically exhibit anoptimal concentration at which cell proliferation is maximal.

The biological activity of HBGF-1 in the presence of thrombin andheparin was also measured by observing endothelial cell proliferation.The surfaces of petri dishes were impregnated with the HBGF-1supplemented FG. A shallow layer of endothelial cells was added and thenumber of cells was measured. Over time the number of cells increased.In addition, the cells appeared to be organizing into vessels.

Therefore, HBGF-1 retains its biological activities in FG that includesheparin, which protects HBGF-1 from the degradative activity of thrombinand may also potentiate the biological activity of the HBGF-1 in thegrowth factor-supplemented FG.

EXAMPLE 4 HBGF-1 Diffusion from a FG Clot

A FG clot was formed in a 5 ml plastic test tube by mixing 0.3 ml of thefibrinogen complex containing 10 U/ml heparin and thrombin and 40 mMCaCl₂. Four test tubes were set up as follows:

(A) 0.5 U/ml thrombin and 10 μg/ml HBGF-1;

(B) 0.5 U/ml thrombin and 50 μg/ml HBGF-1;

(C) 5 U/ml thrombin and 10 μg/ml HBGF-1; and

(D) 5 U/ml thrombin and 50 μg/ml HBGF-1.

Each clot was covered with 0.2 M histidine buffer, pH 7.3. Thirty μlsamples of the overlying buffer were removed from each tube every twohours and were run on a western blot.

The results of the experiment demonstrated that HBGF-1 diffusion out ofthe clot is a function of time and its concentration in the clot, andthat the concentration of thrombin in the clot does not affect the rateat which HBGF-1 is released from the clot.

EXAMPLE 5 The Behavior of Human Umbilical Vein Endothelial Cells inGrowth Factor-Supplemented FG: the Effect of Wild Type and Mutant FGF-1

To study the in vitro effects of acidic fibroblast growth factor(FGF-1)-supplemented FG on human endothelial cells, suspensions of thesecells were added to 10 cm diameter petri dishes that contained evenlyspread layers of 2.5 ml of FG containing approximately 9 mg offibrinogen per ml and 0.25 NIH units of thrombin per ml. The FG wassupplemented in the following ways:

(A) No added growth factor;

(B) Supplemented with 100 ng/ml of active, wild-type FGF-1;

(C) Supplemented with 100 ng/ml of inactive, mutant FGF-1; or

(D) Supplemented with 10 ng/ml of active, wild-type FGF-1.

The cells seeded onto the FG layer were maintained for 7 days in DMEMcontaining 10% fetal bovine serum (FBS).

The cells became elongated and proliferated efficiently when in contactwith FG supplemented with biologically active FGF-1 (FIGS. 3 and 4). Incontact with unsupplemented FG (FIG. 5) or with FG supplemented withbiologically inactive mutant FGF-1 (FIG. 6), the cells become elongatedbut proliferated relatively slowly.

EXAMPLE 6 The Behavior of Human Umbilical Vein Endothelial Cells inFGF-1-Supplemented FG

To study their growth, human umbilical endothelial cells, 10⁵ or morecells per ml, were embedded in FG, the protein concentration of whichwas 4 mg/ml. The concentration of thrombin in the FG was adjusted to 0.6NIH U/ml. The culture medium used in all of the experiments was M199(Sigma Chemical Co., St. Louis, Mo.) supplemented with 10% fetal bovineserum, 10 μg/ml streptomycin, 100 U/ml penicillin, 1 ng/ml FGF-1 and 10U/ml heparin.

Within 24 hours in FG the cells became elongated, multipodial and formeda cellular network when they came in contact with each other (FIG. 7).This growth continued for at least 5 days. FIG. 8 shows this situationat 48 hours.

As a control, an identical cell suspension was cultured on a surfacecoated with fibronectin at 10 μg/cm². Control cells acquired acobblestone shape and maintained this morphology for at least 5 days.FIGS. 9 and 10 show this situation at 24 and 48 hours, respectively.

EXAMPLE 7 The Behavior of PMEXNEO-3T3-2.2 Cells in FG

PMEXNEO-3T3-2.2 cells are fibroblast cells that contain a modifiedgenome with the potential to express genetically engineered proteins(Forough et al., J. Biol. Chem. 268:2960–2968 (1993)). To determine thebehavior of these cells in FG, 10⁵ cells per well were cultured underthree conditions: (1) embedded in FG; (2) on the surface of FG; and (3)in the absence of FG (controls). The experiments were carried out induplicate in 24-well plates in DMEM media (Sigma Chemical Co., St.Louis, Mo.) supplemented with 10% FBS. The FG protein concentration was4 mg/ml. In identical experiments the medium was supplemented with 1.5%FBS was used as negative controls.

In the presence of media supplemented with 10% FBS, the cells in all 3groups grew and became confluent. In the negative control experiments inwhich the media was supplemented with 1.5% FBS, the cells grew andsurvived for at least five days in the presence of FG, but not withoutit. However, their growth was faster in FG supplemented with 10% FBSthan in that supplemented with 1.5% FBS. In the absence of FG, in themedia supplemented with 1.5% FBS, the cells died within 48 hours. Thecriteria for survival was the ability of the tested cells to proliferateupon transfer to fresh media supplemented with 10% FBS.

EXAMPLE 8 The Endothelialization of Expanded PTFE Vascular Grafts byHBGF-1 Pretreatment

Two studies demonstrated that pretreatment of blood-contactingbiomaterials with endothelial cell (EC) mitogens enhancedendothelialization. The first study examined the in vivo washoutcharacteristics of HBGF-1-supplemented FG suspension applied to expandedPTFE grafts implanted into rabbit aortas. In the second study similargrafts were implanted into the aortaileac position in dogs. HBGF-1, anangiogenic factor, was used in studies. Other growth factors such as aFGF, FGF-4 and/or OP-1 can also be used as a supplement(s) for thevascular grafts.

A. Washout Study

In general, the modified FG was sterilely prepared by addingapproximately 1 ng/cm² area of the inner and outer graft surfaces ofhuman recombinant ¹²⁵I-HBGF-1, 20 μg/cm² porcine intestinal mucosalheparin, and 2.86 mg/cm² fibrinogen to 2.86×10⁻² U/cm² reconstituted,commercially available, human thrombin (1000 U/ml) to inducepolymerization.

The ¹²⁵I-HBGF-1 was specifically prepared as follows. Fibrinogen wasreconstituted by adding 500 mg of fibrinogen into 25 ml of PBS toproduce a fibrinogen concentration of 20 mg/ml of PBS. Three ml of thissolution which contained 60 mg fibrinogen were placed into 12 Eppendorfplastic tubes and maintained at −70° C. Each of these aliquots was usedindividually.

The thrombin was reconstituted by diluting a commercially availablepreparation thereof (Armour Pharmaceutical Co., Kankakee, Ill.) at aconcentration of 1000 U/ml by a factor of 1:10 in sterile solution toproduce a concentration of 100 U/ml. This thrombin solution was againdiluted 1:10 to produce a solution of 10 U/ml.

The bovine heparin (Upjohn, Kalamazoo, Mich.) was reconstituted bydiluting the preparation at a concentration of 1000 U/ml by a factor of1:1000 using normal saline.

One and 48/100 (1.48) ml of the reconstituted fibrinogen, 63 μL of thereconstituted heparin, plus 15.66/L of ¹²⁵I-HBGF-1 were mixed in a glassscintillation tube. This mixture was then aspirated into a 3 ml plasticsyringe. Five ml of the reconstituted thrombin was placed into a glassscintillation tube.

One end of the expanded PTFE graft was placed over a plastic 3-waystopcock nozzle and was secured there with a 2-0 silk tie. The PTFE wasthen encircled with a 3×3 cm square of Parafilm™ which was then crimpedthere with a straight hemostat to establish a watertight seal. A second2-0 silk tie was positioned over the parafilm adjacent to the stopcockto form another seal. A straight hemostat was then used to clamp thedistal 2 mm of the PTFE/parafilm to seal this end.

Equal volumes of fibrinogen and thrombin solution prepared as describedabove were mixed and allowed to react for approximately 30 seconds whichis when polymerization occurs. The thrombin-polymerized fibrin is thenopaque. (This time factor is approximate and varies from one thrombinlot to another. The appropriate length of time to polymerization can bedetermined by viewing the opacity of the mixture). The fibrin/thrombinmixture was aspirated into a one cc syringe. (NOTE: The volume of thisgraft was 0.42 ml. For a graft with a larger volume one needs to use alarger syringe.) The syringe was attached to the stopcock and themixture was injected by hand over a period of 5 seconds until the liquidwas seen to “sweat” through the PTFE interstices and filled the spacebetween the PTFE and the Parafilm™. The 3-way stopcock was closed to thePTFE graft for 3 minutes and a scalpel blade was used to cut theligature at the end of the PTFE over the stopcock. The PTFEgraft/parafilm was removed from the stopcock and a hemostat was used toremove the PTFE from the parafilm envelope. To clear residual growthfactor-supplemented FG from the graft lumen, a number 3 embolectomycatheter was passed through the graft five times until the graft lumenwas completely clear. The growth factor-supplemented FG-treated PTFEgraft was allowed to dry overnight for about 12 hours under a laminarflow hood. The treated graft was then ready for implantation.

Alternatively, this HBGF-supplemented FG was pressure perfused into a 34mm (24 mm+5 mm at each end)×4 mm (internal diameter) thin-walled,expanded PTFE graft thereby coating the graft's luminal surface andextending through the nodes to the graft's outer surfaces. The lumen ofthe graft was cleared as stated above. These grafts were then interposedinto the infrarenal abdominal aortas of 24, 3–5 Kg New Zealand whiterabbits. In the first study, the animals were sacrificed and specimenswere explanted at 0 time (to correct for losses due to surgicalmanipulation) and after 5, 30, and 60 min, and 1, 7, 14, and 30 days.Residual radioactivity was determined by gamma counting. Remaining¹²⁵I-HBGF-1, corrected for spontaneous decay, is expressed as apercentage of the zero time value.

The washout of ¹²⁵I-HBGF-1 followed classic kinetics with a rapidinitial loss with the reestablishment of circulation (%/min=−24.1between 5 and 60 minutes) followed by a slow loss after 1 hr(%/min=−0.03) with 13.4%±6.9% remaining after 1 week and 3.8%±1.1%remaining after 30 days.

B. In Vivo Endothelialization Study

The second study evaluated the effects of the appliedHBGF-1-supplemented FG suspension on: the rate of endothelialization ofwidely expanded 60 μm internodal distance expanded PTFE grafts implantedinto canine aorta-iliac positions; the proliferative activity of theseendothelial cells as a function of time; and the relative contributionsof the HBGF-1 and the FG in stimulating the observed endothelial cellproliferation. Three groups of 50×4 mm non-reinforced expanded PTFEgrafts were implanted in the aortailiac position of 12 dogs. Group 1(n=6) contained 20 μg/cm² heparin, 2.86 mg/cm² fibrinogen and 2.86×10⁻²U/cm² of human thrombin plus 1 ng/cm² of HBGF-1. Group 2 (n=3) containedthe same FG without HBGF-1. Group 3 (n=3) consisted of identical butuntreated control grafts. Tritiated thymidine (³H-TdR; 0.5 μCi/kg) wasinjected in 10 hours before explantation. Grafts were explanted at 7 and28 days for light and electron microscopy, Factor VIIIimmunohistochemistry, and en face autoradiography for endothelial cellproliferation in random high power fields. Each graft was viewed bythree observers who did not know from which treatment group the graftcame. Differences in endothelial cell proliferation were statisticallyanalyzed by two-way ANOVA and independent t-tests.

At 7 days 33% of both the FG and HBGF-1-supplemented FG graftsdemonstrated non-contiguous foci of endothelial cells (FIG. 11). Thesurface of the control grafts remained a fibrin coagulum. At 28 days,every HBGF-1-supplemented FG showed extensive capillary ingrowth andconfluent endothelialized blood contacting surfaces, which were not seenin any specimen of the other two groups (FIGS. 11 and 12). FIG. 12demonstrates that untreated grafts at 28 days had few visibleendothelial cells on their surface (Panel G). Grafts treated with FGalone had about 33% of their surface covered with endothelial cellsindicating that FG treatment alone encouraged some reendothelialization(Panel H). However, grafts treated with FG supplemented with HBGF-1(Panel I) appeared to be completely (>95%) covered with endothelialcells which display the characteristic cobblestone morphology ofendothelial cells. Thus, the combination of growth factors delivered byFG was able to encourage essentially the complete covering of thevascular graft with a non-thrombogenic endothelial cell lining. En faceautoradiography revealed a statistically significant increase (p<0.05)in ³H-TdR incorporation into the DNA of endothelial cells in theHBGF-1-supplemented FG grafts at 28 days vs. all other groups both as afunction of time and of graft treatment.

These data demonstrate that pressure perfusion of an HBGF-1-supplementedFG suspension into 60μ internodal distance expanded PTFE grafts promotesendothelialization via capillary ingrowth and increased endothelial cellproliferation.

These studies demonstrate enhanced spontaneous re-endothelialization ofsmall diameter vascular grafts, and also a method for stimulating a morerapid confluence of transplanted endothelial cells.

EXAMPLE 9 Delivery of Tributyrin from Fibrin Sealant

The induction of endothelialization of artificial vascular grafts byFGF-1 delivered in fibrin sealant represents an important therapeuticapplication of the use of supplemented fibrin sealant as a deliveryvehicle. Hyperproliferation of smooth muscle cells in arterial walls isa significant component of arteriosclerosis, and in restenosis followingangioplasty (Cercek et al., Amer. J. Cardiol. 68:24C (1991)). Therefore,delivery of an anti-proliferative or differentiating agent suitable forintravascular treatment from a supplemented fibrin sealant deliverysystem was considered to prevent or treat this condition.

In choosing an agent to prevent smooth muscle cell hyperproliferation, adrug with extremely low toxicity was selected as it was important not toinduce cell damage that might exacerbate the underlying condition.Butyric acid has been shown to prevent the hyperproliferation ofretinoblastoma cells (Kyritsis et al., Anticancer Res. 6:465 (1986)),Swiss 3T3 cells (Toscani et al., J. Biol. Chem. 265:5722 (1990)) andother cell types (Prasad et al., Life Sci. 27:1351 (1980)) by inducing adifferentiation program.

An induced prevention of hyperproliferation also has been achieved insmooth muscle cells by a related compound, tributyrin. This effect onsmooth muscle cells requires a concentration of tributyrin that is closeto saturation, making systemic therapy difficult. Therefore, thefollowing experiment was conducted to demonstrate the efficacy ofdelivering tributyrin directly to the lesion from a supplemented fibrinsealant composition.

Tributyrin was mixed with thrombin, which was then mixed with fibrinogento form a fibrin sealant matrix. The supplemented fibrin sealant wasplaced into 24-well culture plates. Culture medium (2 ml) was thenplaced in wells containing the tributyrin supplemented fibrin sealant,and these were incubated at 37° C. The medium from a new set of threewells was harvested daily, and the supernatant used to cultureproliferating smooth muscle cells (10,000 rat or rabbit smooth musclecells per well, which had been allowed to attach overnight). Afterincubation for two days (48 hours), the number of cells in each smoothmuscle cell culture was measured using the MTS assay (a bioreduction ofthe tetrazolium compound MTS (Promega, Madison, Wis.) into a solubleformazan chromatophore detected by spectrophotometry at 490 nm.)

As shown in FIG. 13, the medium harvested from wells containing fibrinsealant alone supported the growth of the smooth muscle cells, while themedium from wells with fibrin sealant containing tributyrinsignificantly inhibited smooth muscle cell proliferation. As the numberof days of tributyrin diffusion into the medium increased, the degree ofinhibition increased. These results indicated that a cell regulatorydrug, tributyrin, can be delivered from fibrin sealant for extendedperiods and that it retains the sustained ability to inhibit theproliferation of a specific cell type.

EXAMPLE 10 Formulation and Delivery of TGF-β2 from Fibrin Sealant

Fibrinogen and thrombin were prepared per instruction of the AmericanRed Cross, Rockville, Md. Upon reconstitution, the protein concentrationof the Topical Fibrinogen Complex, (TFC) was 120 mg/ml (the standardformulation for hemostasis). The human thrombin was reconstituted with40 mM CaCl2 to yield a solution at 300 units/ml.

To evaluate the compatibility of transforming growth factor β2 (TGF-β2)in Topical Fibrinogen Complex, TGF-β2 (purified recombinant humanprotein provided by Genzyme Corp., Framingham, Mass.) was spiked intoTFC at 10 and 1 μg/ml. Samples were incubated for two weeks at 2–8° C.TGF-β2 was extracted for analysis by passing the gel-like materialthrough a narrow bore stopcock connected to two syringes. The ELISA dataindicated full recovery of TGF-β2 from the TFC. Analysis in the in vitrobio-assay indicated that the extract was bioactive.

TGF-β2 was then spiked into the TFC solution at a concentration of 1μg/ml or 100 ng/ml. 50 μl aliquots were placed into sterile test tubesand 50 μl of the thrombin solution was added to form the fibrin clot.Clot formation occurred within a few seconds. These samples were allowedto sit overnight at 2–8° C. Test sample tubes were then overlaid with400 μl of PBS/0.1% human serum albumin pH 7.0, with or without 10 μg/mlplasmin. The test samples were incubated for two days at 37° C. toevaluate the release and recovery of the TGF-β2. Complete resolution ofthe clot was observed in the plasmin treated samples. The clot remainedintact in the non-plasmin treated samples. The diffusion supernatant wasanalyzed by ELISA. The data are summarized in Table 1.

TABLE 1 % Recovery in Diffusion Supernatant (by ELISA) TGF-β2Concentration With Without in Fibrin Clot Plasmin Plasmin 500 ng/ml 100%2.5%  50 ng/ml 100% (not detectable) Theoretical concentrations ofcomponents in the final clot based on dilution: TFC protein = 60 mg/ml;thrombin activity = 150 units/ml; TGF-β2 = 500 ng/ml or 50 ng/ml.

The data indicate not only that TGF-β2 is stable in TFC, but that thedelivery of TGF-β2 from fibrin sealant by diffusion can be sustained inlow amounts. Moreover, the release of TGF-β2 from fibrin sealantrequires dissolution of the fibrin clot by plasmin indicating that invivo delivery of TGF-β2 from the supplemented tissue sealant compositionwould be mediated by resolution of the fibrin clot. Thus, the mechanismof delivery from the TGF-β2 supplemented tissue sealant composition isreadily distinguished from simple diffusion kinetics.

EXAMPLE 11 The Preparation of a Platelet-Derived Extract for Use with FG

Plasma reduced platelets were prepared and pelleted. The supernatantplasma was removed. The pelleted platelets were washed, suspended inbuffer containing 50 mM histidine and 0.15 M sodium chloride at pH 6.5,and treated with bovine thrombin. After treatment, the supernatant wascollected by centrifugation and aliquots were frozen at −80° C. Theextract was thawed and mixed with FG or other TSs.

The platelet extract obtained in this manner was biologically activesince it increased the incorporation of radioactive labeled thymidineinto the DNA of proliferating NIH3T3 fibroblasts compared to thecontrols.

To evaluate the effect of platelet extract on wound healing, experimentsidentical to those carried out below in Example 12 with HBGF-1β werecarried out with platelet extract in diabetic mice. From the results ofthese experiments is clear that, given the low concentration of growthfactors in the platelet extract, a dose larger than 100 μg of plateletextract protein per wound needs to be used to promote wound healing.

EXAMPLE 12 The Effect of FG on Skin Wound Healing In Vivo

A. Unsupplemented FG

Animals

Female C57BL/K_(S)J-db/db mice were obtained from Jackson Laboratories(Bar Harbor, Me.) and were 8 to 12 weeks old at the start of theexperiment. They were housed in separate cages after surgery in ananimal care facility.

These mice are used as a model of impaired wound healing in diabetichumans because the metabolic abnormalities seen in these mice aresimilar to those found in human diabetics. In addition, the healingimpairment characterized by markedly delayed cellular infiltration,granulation tissue formation, and time required for wound closuresuggest that healing in this mouse model may be relevant to the healingimpairment seen in human diabetes.

Fibrin Sealant

The concentrated topical fibrinogen complex (TFC) used in this study wasproduced from fresh frozen pooled human plasma. The TFC product(American Red Cross—Baxter Hyland Division, Los Angeles, Calif.) wassupplied in lyophilized form. After reconstitution with 3.3 ml ofsterile water, the protein characteristics of the TFC solution used inthis study were: total protein, 120 mg/ml; fibrinogen, 90 mg/ml;fibronectin, 13.5 mg/ml; Factor XIII, 17 U/ml; and plasminogen, 2.2μg/ml.

Topical bovine thrombin (5000-unit vial, Armour Pharmaceutical Co.,Kankakee, Ill.) was reconstituted with 5 ml sterile water and wasserially diluted in 80 mM calcium chloride solution (American ReagentLaboratories, Shirley, N.Y.) to a concentration of 15 U/ml.

Equal volumes of TFC and reconstituted thrombin were mixed to produceFG. In order to fill a round 6-mm-diameter full thickness wound, 0.015ml of TFC was mixed with 0.015 ml of thrombin. The FG that was producedhad a protein concentration of approximately 60 mg/ml.

A diluted FG with a protein concentration of approximately 1 mg/ml wasalso used.

Surgery

The mice were anesthetized with a mixture consisting of 7 ml ketaminehydrochloride (100 mg/ml; Ketaset, Aveco Co., Inc., Fort Dodge, Iowa), 3ml xylazine (20 mg/ml; Rompun, Mobey Corp., Shawnee, Kans.), and 20 mlphysiological saline, at a dose of 0.1 ml per 100 g body wt,administered intramuscularly. The dorsal hair was clipped, and the skinwas washed with povidone-iodine solution and wiped with 70% alcoholsolution. Two full-thickness, round surgical wounds (6 mm diameter) weremade on the lower back of the mouse, one on each side, equidistant fromthe midline. The medial edges of the two wounds were separated by amargin of at least 1.5 cm of unwounded skin.

Immediately after the wounding had been performed, FG and/or a dressingwas placed over the designated wound. The dressing was a transparentsemipermeable adhesive polyurethane dressing (Opsite™, Smith and Nephew,Massillon, Ohio). Tincture of Benzoin compound (Paddock Laboratories,Minneapolis, Minn.) was applied at the periphery of the wound area priorto application of the dressing. There was a margin of at least 0.5 cm ofskin surrounding the wound edge over which no tincture of benzoin wasapplied to avoid the possible inflammatory effects of benzoin on the rawwound. No further treatments were applied to the wound for the durationof the experiment.

Treatment Groups

The mice were divided into 4 treatment groups, with each mouse servingas its own control:

Group I: The wound on one side of the animal was treated with FG (60mg/ml) while the contralateral wound received no treatment. Both woundswere covered with Opsite™.

Group II: Diluted FG (1.0 mg/ml) was topically applied to the wound onone side while the contralateral wound received no treatment. Bothwounds were covered with Opsite™.

Group III: FG (60 mg/ml) was topically applied over both wounds. Thewound on one side was left uncovered while the contralateral wound wascovered with Opsite™.

Group IV: No topical treatment was applied over the wounds. The wound onone side of the animal was left uncovered while the wound on thecontralateral side was covered with Opsite™.

Wound Analysis

The animals were euthanized on Day 9 of the experiment. The wounds wereexcised down to the muscle layer, including a margin of 0.5 mm ofunwounded skin, and were placed in buffered 10% formalin solution. Thespecimens were submitted to a histology laboratory for processing.Specimens were embedded in paraffin, and the midportion of the wound wascut in 5-μm sections. The slides were stained with hematoxylin andeosin, or with Masson's trichrome for histologic analysis.

Each slide was given a histological score ranging from 1 to 15, with 1corresponding to no healing and 15 corresponding to a scar withorganized collagen fibers (Table 2). The scoring scale was based onscales used by previous investigators. The criteria used previously weremodified and were further defined to more precisely reflect the extentof: reepithelialization, degree of cellular invasion, granulation tissueformation, collagen deposition, vascularity, and wound contraction. Thehistologic score was assigned

TABLE 2 Criteria for Scoring of Histologic Sections Score Criteria  1–3Epithelialization None to very minimal Cellular content None to veryminimal (mainly inflammatory cells) Granulation tissue None to sparseamount at wound edges Collagen deposition None Vascularity None  4–6Epithelialization Minimal (less than half of wound dia- meter) tomoderate (more than half of wound diameter) Cellular contentPredominantly inflammatory cells, few fibroblasts Granulation tissueNone to thin layer at wound center, thicker at wound edges Collagendeposition Few collagen fibers Vascularity Few capillaries  7–9Epithelialization Completely epithelialized; thin layer Cellular contentMore fibroblasts, still with inflammatory cells Granulation tissue 7,sparse at wound center; mainly adipose tissue underneath epithelium 8,thin layer at wound center; few collagen fibers 9, thicker layer; morecollagen 10–12 Epithelialization Thicker epithelial layer Cellularcontent Predominantly fibroblasts Granulation tissue Uniformly thickCollagen deposition Moderate to extensive collagen deposited, but lessmature when compared to collagen of unwounded skin margin VascularityModerate to extensive neovascularization 13–15 Epithelialization Thickepithelium Cellular content Fewer number of fibroblasts in dermisGranulation tissue Uniformly thick Dense, organized, oriented collagenfibers Few well-defined capillary systemsseparately by at least three analysts. The code describing the woundtreatment was broken after the scoring was completed by all observers.Statistical Analysis

The values of the histological scores of the analysts were averaged andwere expressed as the mean±standard error of the mean.

The paired t test was used for comparison of paired means in thedifferent treatment groups. The analyses were performed using the RS/1Release 3.0 statistical software package (BBN Software ProductsCorporation).

The sample mean differences were tested for analysis of variance usingthe Statistical Analysis Software (SAS) System.

Results

The Effect of FG on Wound Closure (Group I)

In Group I both wounds on each mouse were covered with Opsite™. Underthese conditions, the topical application of FG with a proteinconcentration of 60 mg/ml to only one side of the animal resulted instatistically lower mean histological scores (3.06) for the FG sidecompared to the untreated wounds (5.26) (P<0.005) (Table 3).

TABLE 3 The Effect of FG (60 mg/ml) on Wound Closure (Group I) TreatmentHistologic score N FG + Opsite ™ 3.06 ± 0.7  15 Opsite ™ alone 5.26 ±2.21 15The Effect of Dilute FG on Wound Closure (Group II)

In this group, both paired wounds which were covered with Opsite™,topical application of dilute FG (protein concentration of 1 mg/ml)resulted in a mean histological score (4.0) that was not statisticallydifferent from that for untreated wounds (4.36) (P=0.17) (Table 4).

TABLE 4 The Effect of Dilute FG (1 mg/ml) on Wound Closure (Group II)Treatment Histologic score N Dilute FG + Opsite ™ 4.00 ± 0.77 11Opsite ™ alone 4.36 ± 0.67 11The Effect of Opsite™ on FG-Treated Wounds (Group III)

In this group of paired wounds both treated with FG with a proteinconcentration of 60 mg/ml, the application of Opsite™ to one sideresulted in a mean histological score (4.2) which was not statisticallydifferent from that for wounds which were left uncovered (4.93) (P=0.11)(Table 5).

TABLE 5 The Effect of Opsite ™ on Paired Wounds Treated with FG (GroupIII) Treatment Histologic score N Opsite ™ + FG 4.20 ± 1.93 15 FG but noOpsite ™ 4.93 ± 1.09 15Effect of Opsite™ on the Closure of Paired Untreated Wounds (Group IV)

In this group of paired wounds which did not receive topical treatmentof FG, application of Opsite™ to one side resulted in a significantlylower mean histological score (4.92), as compared to that for woundswhich were left uncovered (6.31) (P<0.0005) (Table 6).

ANOVA of the treatment effects on sample mean differences wassignificant at <0.0001.

TABLE 6 The Effect of Opsite ™ on the Closure of Paired Untreated OpenWounds (Group IV) Treatment Histologic score N Opsite ™ (no FG) 4.92 ±1.26 13 No Opsite ™ (no FG) 6.31 ± 1.25 13Discussion

The results of this study indicated that in mice (1) when applied overopen wounds, FG at a concentration formulated for hemostasis (60 mg/ml)resulted in lower histological scores at Day 9 which indicated slowerrates of wound healing compared to that of untreated wounds; (2)dilution of the FG protein concentration to 1 mg/ml resulted in a higherhistological score at Day 9 which indicated a faster rate of woundhealing; and (3) application of a semipermeable dressing (Opsite™) perse significantly retarded wound closure in this animal model by itself.

The total protein concentration of FG is an important variable whencomparing the results of studies using FG. Beneficial effects of fibrinin promoting wound healing and tissue repair have been reported, butlower concentrations of fibrinogen have been used in the present studiesthan is commonly found in commercial preparations.

FG at a concentration of 60 mg/ml delayed wound closure (Group I). Thetotal protein concentration of FG which is commercially available inEurope, after mixture of the fibrinogen and thrombin components, is 37.5to 57.5 mg/ml. These data indicate that FG as presently formulated forhemostatic and adhesive indications retards healing when applied to openskin wounds. This effect may be due to (1) mechanical obstruction to themigration or proliferation of cellular elements that activelyparticipate in the wound healing process, (2) mechanical inhibition ofwound contraction or (3) a chemical inhibitory effect of one or more FGcomponents on wound healing. Mechanical obstruction and inhibition ofwound closure may be the more likely explanation, since at Day 9 thereis persistence of a solid fibrinogen-based clot on the wound surface.

In order to help determine if this was the cause, the total proteinconcentration of FG was diluted to 1 mg/ml. Topical application of thisdilute FG resulted in a histological score that was not significantlydifferent from that for untreated wounds (Group II), suggesting thatlower total protein concentrations do not significantly inhibit thewound healing process.

It is also worth noting that the mean histological score for coveredwounds treated with the same concentration of FG (60 mg/ml) butbelonging to different treatment groups (Groups I and III) hadsignificantly different values (3.06 for Group I vs. 4.2 for Group III).These data demonstrated that animal to animal variation makes itdifficult to derive definitive conclusions from different animalssubjected to the same treatment variables because some animals may healfaster or slower than the others despite receiving the same treatment.This is reflected in the range of standard errors for the mean scores.For this reason each animal served as its own control, e.g. wounds inthe same animal were compared to each other. By having the controlwounds in the same animal as the test wounds, the effects of interanimalvariability was minimized. These data also show that an adhesivedressing such as Opsite™ significantly delayed wound closure. It shouldbe noted, however, that in partial thickness skin wounds in pigs theprotein concentration of the FG does not appear to be related to therate of wound healing.

B. Growth Factor-Supplemented FG on Wound Healing In Vivo.

The effect of HBGF-1B growth factor-supplemented FG on the rate of woundrepair in diabetic mice was assessed. The methods used in thisexperiment were similar to those just described above. Two 6 mmfull-thickness skin biopsies on the dorsal part of each of 6 test micewere filled with FG to which 5 μg of HBGF-1β had been added. Identicalbiopsies in six mice were left untreated, and in six control mice werefilled with unsupplemented FG. After 9 days, all of the mice weresacrificed and histological preparations of 5 micron thick slices fromeach of the wounds and surrounding skin were prepared and stained withhematoxylin and eosin.

The extent of wound repair in each sample, which was not identified asto the treatment group from which it came, was “blindly” evaluated byeach of three trained analysts, who assessed collagen deposition,reepithelialization, thickness of the granulation tissue and the densityof inflammatory cells, fibroblasts and capillaries. Each sample wasscored from 1 to 15, ranging from no to complete repair. The samplesfrom the wounds treated with unsupplemented FG were consistently giventhe lowest scores and those from the untreated wounds or wounds treatedwith the growth factor-supplemented FG were given the highest scores.

EXAMPLE 13 FG as a Delivery Vehicle of Osteoinductive Substances In Vivo

Fibrin Sealant

Concentrated human TFC (Baxter Hyland Division, San Pedro, Calif.) andhuman thrombin (Baxter Hyland Division, Glendale, Calif.) were producedfor the American Red Cross from screened fresh frozen pooled humanplasma. Both components underwent viral inactivation using the solventdetergent method (New York Blood Center) during their production andwere supplied in lyophilized form. After reconstitution with 3.3 ml ofsterile water, the protein characteristics of the TFC solution were:total protein=120 mg/ml; fibrinogen=90 mg/ml; fibronectin=13.5 mg/ml;Factor XIII=17 U/ml; and plasminogen=2.2 μg/ml.

Human thrombin (1000 U vial) was reconstituted with 3.3 ml sterilewater, and was serially diluted in 40 mM calcium chloride solution(American Regent Laboratories, Shirley, N.Y.) to a concentration of 15U/ml. Human thrombin was used for preparing disks implanted which wereonto calvarial defects.

Topical bovine thrombin (5000 U vial, Armour Pharmaceutical Co.,Kankakee, Ill.) was reconstituted with 5 ml sterile water, and wasserially diluted in 40 mM calcium chloride solution to a concentrationof 15 U/ml. Bovine thrombin was used for preparing implants forintramuscular bioassay.

In practicing this embodiment of this invention the fibrinogen should bepresent at a concentration of 1 to 120 mg/ml FG, more preferably from 3to 60 mg/ml FG, most preferably from 10 to 30 mg/ml FG. DBM should bepresent at an approximate concentration of about 1 to 1000 mg/ml FG,more preferably from 50 to 500 mg/ml FG, most preferably from 300–500mg/ml FG. The particle size of demineralized bone powder should be from0.01 to 1000 microns, preferably from 20–500 microns and most preferablyfrom 70–250 microns. The osteoinductive growth factor(s) or BMPs shouldbe present at a concentration(s) of about 1 to 100 μg/ml wherein theconcentration(s) is effective to accomplish its desired purpose. Growthfactors which may be used as osteoinductive substances in thisembodiment include, but are not limited to: osteogenin (BMP3); BMP-2;OP-1; HBGF-1; HBGF-2; BMP 2A, 2B and 7; FGF-1; FGF-4; and TGF-β. Inaddition, drugs, such as antibiotics, can be used to supplement the TSfor use in bone repair.

Implant Preparation

Rat DBM was prepared as follows. The epiphyses of the long bones of ratswere removed leaving only the diaphyses behind. The diaphyses weresplit, if necessary, and the bone marrow was then thoroughly flushedwith deionized water (Milli-Q Water Purification System™, MilliporeCorporation, Bedford, Mass.). The diaphyses were then washed at roomtemperature. At 4° C., 1000 mls of deionized water was added to 100 g ofbone. The mixture was stirred for 30 minutes and the water was decanted.This step was repeated for two hours.

At 4° C., one liter of cold absolute ethanol (Quantum ChemicalCorporation, U.S.I. Division, Tuscola, Ill.) was added for every 100 gof bone. After stirring for 15 minutes, the ethanol was decanted. Thiswas repeated four times for a total of one hour's duration.

Under a fume hood, 500 ml of diethyl ether (Mallinckrodt SpecialityChemicals, Paris, Ky.) was added to the bone to cover it. This wasstirred gently for 15 minutes and the ether was then decanted. Anadditional 500 mls of ether was added to the bone and the mixture wasstirred for 15 minutes. The ether was again decanted. The bone was leftunder the fume hood for the evaporation of the ether to occur. Defattedbone can be stored indefinitely in an ultralow freezer (−135° C.).

The bone was then milled to make bone powder. The powder was sieved and74 to 420 micron size particles were collected.

Ten gram aliquots of the bone powder were placed in 250 ml centrifugebottles. Eighty mls of 0.5 N HCl was added to each bottle slowly inorder to avoid frothing. The contents of each bottle were then stirredgently. After 15 minutes, an additional 100 mls more of 0.5 N HCl wasadded to each bottle over the course of 10 minutes. The bottles werethen stirred gently for an additional 35 minutes. The total time thatthe powder was in the HCl did not exceed one hour.

Each mixture was then spun in a centrifuge at 3000 rpm at 4° C. for 15minutes. The pH of the supernatant was then checked. If the pH wasgreater than 2, the supernatant was poured down a chemical sink withoutdisturbing the pellet(s). If the pH of the supernatant was less than 2,the supernatant was poured off into a hazardous waste container. If thepellet(s) were loose, the centrifuge time was increased to 30 minutes.These steps were repeated until the pH of the supernatant was equal to0.5 N HCl.

The pellets were then washed with 180 mls of deionized water by stirringto produce an even suspension. The suspension was then centrifuged foran additional 15 minutes. The supernatant was then decanted as before.The washing was repeated until the pH of the supernatant equaled the pHof the deionized water.

The pellets were then frozen at −180° C. in a freezer. They were thenlyophilized using standard procedures.

Disk-shaped implants 1 mm thick and 8 mm in diameter were produced usinga 4-piece aluminum mold (FIG. 14). Twenty-five mg of rat DBM powder wasadded into the mold chamber. Thirty μl f TFC was then pipetted onto theDBM and mixed until the DBM had absorbed all of the solution. Theconcentrations of TFC which were used were 10, 20, 40, 80, or 120 mg/ml.Thirty μl of thrombin solution (15 U/ml in 40 mM calcium chloridesolution) was then added to the DBM-TFC complex, was mixed, and wascompressed into a disk-shape using a piston-shaped lid. It wasdetermined that 25 mg of DBM powder had a volume of 20 μl . After DBMhad been added to the FG, the final protein concentrations were asfollows:

TABLE 7 TFC Thrombin DBM FG, Total protein (mg/ml) (μ/ml) (mg) conc.(mg/ml) 120 15 25 45 80 15 25 30 40 15 25 15 20 15 25 8 10 15 25 4

Disk implants composed of DBM alone or FG alone (4, 8, 15 and 45 mg/mltotal protein concentrations) were likewise made using the same mold.

Fifty mg of DBM was poured into an aluminum mold, to which 60 μl of TFCwas then added to the DBM and mixed until fully absorbed. Sixty μl ofthrombin was then added to the DBM-TFC complex, mixed and compressedinto a disk-shape with a diameter of 1 cm and a thickness of 2 mm usinga piston-shaped lid. The disk was then cut manually into the desiredshape (triangle, square or donut).

For the intramuscular bioassay experiment, implants were placed in asterile nylon bag having a mesh size of 70 microns and measuring 1 cm×1cm.

Animals

Male Long-Evans rats were obtained from Charles River Laboratories(Wilmington, Mass.). For the intramuscular bioassay, 28 to 35 day oldrats were used. Three month old rats were used for the craniotomyexperiment.

Surgery

The animals were anesthetized with a mixture consisting of 10 mlketamine hydrochloride (Vetalar, 100 mg/ml, Parke-Davis, Morris Plains,N.J.), 5 ml xylazine (Rompun, 20 mg/ml, Mobay Corporation, Shawnee, KN),and 1 ml physiologic saline (0.9% NaCl), at a dose of 0.1 ml per 100 gmbody weight, administered intramuscularly. The operative site of theanimal was prepped with 70% alcohol solution, followed bypovidone-iodine solution. The surgical procedure was then performedusing aseptic technique.

Intramuscular Bioassay. A midline ventral incision was made and a spacewas created between the pectoralis muscles with blunt dissection. Anylon envelope containing the designated experimental material wasinserted into the intramuscular space and secured with a 3-0 Dexonsuture (FIG. 15). The same procedure was then repeated at thecontralateral side. The skin was then closed with staples. The implantswere harvested after four weeks, were x-rayed and were prepared forhistology.

Disk-shaped implants were placed randomly and consisted of thefollowing: DBM alone (n=12); FG alone at different concentrations (4mg/ml, n=14; 8 mg/ml, n=3; 15 mg/ml, n=3; and 45 mg/ml, n=12), andDBM-FG complex (4 mg/ml, n=12; 8 mg/ml, n=12; 15 mg/ml, n=12; and 45mg/ml, n=12). There were four each of the square-, triangle- anddonut-shaped implants.

Craniotomy Procedure. A linear incision was made from the nasal bone tothe mid-sagittal crest. Soft tissues were reflected gently and theperiosteum was dissected from the craniotomy site (occipital, frontal,parietal bones). An 8-mm craniotomy was prepared with a trephine in aslow-speed rotary handpiece using copious saline irrigation as needed.The calvarial disk was dissected free while avoiding dural perforationsand superior sagittal sinus intrusion. The 8-mm calvarial defect waseither left untreated as control or filled with a 1×8-mm DBM or DBM-FGdisk (FIG. 16). The skin was then closed with skin staples.

Following surgery, each rat was identified by ear punches and returnedto its cage where they were ambulatory within 2–3 hours.

The first set of calvarial implants consisted of DBM alone (25 mg, n=3)or DBM in a FG matrix (15 mg/ml, n=2; 30 mg/ml, n=3; and 45 mg/ml, n=3),and were retrieved after 28 days. The second set of calvarial implantsconsisted of 25 mg DBM in a 30 mg/ml FG matrix and were retrieved atdifferent postoperative times (28 days, n=10; 3 months, n=9; and 4months, n=5).

Retrieval of Implants

At the indicated times, the rats were euthanized in a carbon dioxidechamber. A skin incision was made around the experimental recipient bed(i.e., pectoralis major or calvaria) and the soft tissues were reflectedfrom the recipient beds. In orthotopic sites, the craniotomies with 3–4mm contiguous bone were recovered from the fronto-occipito-parietalcomplex. In heterotopic sites, sharp and blunt dissection was used torecover the implanted nylon envelopes.

Radiography

The implants were radiographed using X-OMATL™ high contrast Kodak x-rayfilm (Eastman Kodak Company, Rochester, N.Y.) in a Minishot BenchtopCabinet x-ray system (TFI Corporation, West Haven, Conn.) at 30 kvp, 3Ma, and 10 seconds. Gray-level densities of intramuscular and craniotomysite radiographs were analyzed using a Cambridge 920 Image AnalysisSystem™ (Cambridge Instruments Limited, Cambridge, England).

Histological Analysis

All retrieved specimens (soft and hard tissues) were immediately placedinto appropriately labeled vials containing preservative solution andwere submitted to a histology laboratory for processing. Histologicspecimens were 4.5 micrometer-thick sections through the coronaldiameter. For each recipient site, one section was prepared withhematoxylin and eosin stain (for photomicrography and examination ofcell and stromal detail) and the other section was prepared with a vonKossa stain.

Results

Radiography of Intramuscular Plants

All DBM disks displayed radio-opaque images. Forty-five out of 48implanted DBM-FG disks (93.75%) were radio-opaque. All DBM-FG disks,regardless of protein concentration (4–45 mg/ml) induced radio-opacity(FIG. 17). Radio-opacity measurements of some DBM disks (FIG. 17) werehigher than DBM-FG disks but the other measurements were well within therange of measurements for DBM-FG disks. Thirty out of 32 FG disks whichwere not supplemented with DBM (93.75%) did not develop radio-opacity.

DBM-FG disks in the form of squares, triangles or donuts were alsomarkedly radio-opaque as compared to FG disks which were notsupplemented with DBM. The original shapes of the implants weregenerally retained.

Histology of Intramuscular Implants

The intramuscular bioassay was positive for DBM and DBM-FG implants, asevidenced by formation of ossicles with a central cavity filled withmarrow and resorption of previously implanted DBM particles.

Radiography of Calvarial Implants

X-rays showed DBM implants in a FG matrix to be generally moreradio-opaque than DBM implants alone or untreated controls. There was nomarked discernible difference between different concentrations of FGused to deliver DBM. The radiographs of untreated 8-mm diameter calvariadefects showed a negligible amount of radio-opacity.

The second set of calvarial implants using DBM in 30 mg/ml FG matrixshowed markedly increased radio-opacity within the craniotomy wounds of3 or 4 month-old calvaria over 28 day calvaria (FIG. 18).

Histology of Calvarial Implants

Non-treated 8 mm craniotomy wounds showed only fibrous connective tissuedeveloping across the craniotomy wound (FIGS. 19A and B). Histology ofDBM implants showed DBM particles to be scattered all over the field.Some DBM particles migrated over and under the edges of host bone (FIG.20). Most DBM particles were, however, within the confines of thecraniotomy wound and were surrounded by loose connective tissue that waswell vascularized. Active resorption of DBM by osteoclasts was noted. Alot of DBM particles were also noted to be populated by live cells. Newosteoid and bone laid down by osteoblasts were quite evident.

The histology of DBM implants in a FG matrix showed DBM particleslocalized within the craniotomy wound, surrounded by much denser andmore cellular connective tissue (FIGS. 19 and 20). Osteoid matrix andbony trabeculae formation were quite evident. More bone marrow was notedto have formed in craniotomy wounds implanted with DBM-FG disks thanwith DBM implants alone. There was also greater neovascularization withDBM-FG disks than with DBM implants alone or untreated controls.Osteoregeneration was evident at all concentrations of FG used todeliver DBM.

Discussion

The natural biocompatibility and biodegradability of FG arecharacteristics that make it an ideal delivery vehicle for DBM and BMPs.FG facilitated the shaping of DBM into the desired form to fill bonydefects, maintained DBM within the defect, and may have been synergisticwith DBM. Furthermore, soft tissue prolapse did not occur and bonycontour was maintained. DBM-supplemented FG possessed an appropriatemicroarchitecture, biodegradation profile and release kinetics tosupport osteoblast recruitment and osteoregeneration.

Overall, the data indicated that DBM delivered in FG at any of thetested FG protein concentrations induced as much bone formation as theDBM did alone. Moreover, when DBM was configured with FG to a particularpre-operative form, the induced bone closely retained the original shapepostoperatively.

Since the shape of the DBM-FG matrix determined the morphology of thenewly formed bone, when possible, the DBM-FG matrix should be made of apredetermined shape. However, the DBM-FG matrix in liquid form can bedelivered or injected into an irregularly shaped defect where it willpolymerize and encourage bone formation in the DBM-FG-filled area.

EXAMPLE 14 The Release of Antibiotics (AB) from FG and IncreasedLongevity of the AB-Supplemented FG

A. Preparation of the AB-FG

1. TET Free Base

Three-and-one-half ml of water for injection was injected into a vial oflyophilized human topical fibrinogen concentrate (TFC), supplied by TheAmerican Red Cross. The protein concentration of the resulting solutionwas approximately 120 mg/ml.

Freeze-dried thrombin concentrate, supplied by The American RedCross/Baxter-Hyland, Inc., Glendale, Calif., was reconstituted with 3.5ml of a 40 mM solution of calcium chloride prepared in water forinjection. The resulting solution contained approximately 250 U/ml.

TET-FG was formulated by mixing the desired weight of TET with 1 ml ofreconstituted TFC solution and with 1 ml of reconstituted thrombinsolution in the presence of injection quality calcium chloride(purchased from American Reagent, Shirley, N.Y.). The TET was in thefree base form and was purchased from Sigma Chemical Company (St. Louis,Mo.). The TET-FG was formed by mixing TFC and thrombin through a Duoflo™dispenser (Hamaedics, Calif.) onto a Millipore membrane in a 12 mmdiameter Millipore culture plate (Millipore Corporation, Bedford,Mass.). The mixture was allowed to set for one hour at 22° C. Six mmdiameter disks containing the TET-FG and the Millipore membrane were cutfrom the latter using a 6 mm punch biopsy. The TET-FG-containing diskswere used for the TET release studies.

The release of TET from the TET-FG into phosphate buffered saline (PBS)or saliva was measured using 24-well cell culture plates (Corning GlassWorks, Corning, N.Y.) under two different sets of conditions. In onecondition, the static mode, 2 ml of PBS or 0.75 ml of saliva wasreplaced daily in the 24-well cell culture plates. In the othercondition, the continuous exchange mode, TET release from the TET-FG wasmeasured with PBS having been exchanged at a rate of approximately 3 mlper day. The samples were stored at −20° C. until analyzed. The salivahad been collected from 10 different people, had been pooled, andclarified by centrifugation at 5000 g. It was then filtered through a0.45 μm pore sized membrane and was stored at 4° C. for daily use.

In order to measure the concentration and biological activity of the TETwhich had been released from the TET-FG disks, the eluted TET was thawedand was analyzed spectrophotometrically at 320 nm and/or biologically bythe inhibition of E. coli growth on agar plates. To calibrate theseassays, standard curves covering TET concentrations of from 0 to 50 and0 to 500 μg/ml, respectively, were used.

2. Ciprofloxacin HCl (CIP)-, Amoxicillin (AMO)- and Metronidazole (MET)Supplemented FG.

FG containing CIP HCl, AMO or MET were prepared as before for TET. Tomonitor the release of these AB from the corresponding AB-FG into theimmediate environment, the AB-FG disks were placed in individual wellsin a 24-well cell culture plate and were covered with 2 ml of PBS thatwas collected, replaced daily and stored at −20° C. as before, untilanalyzed. The concentrations of CIP, AMO and MET in the eluates weremeasured spectrophotometrically at 275, 274 and 320 nm, respectively,and were compared to standard curves containing 0 to 50 ug/ml of thecorresponding AB.

B. Structural Integrity of AB-FG

The maintenance of the structural integrity of the FG and the TET-FGdisks was estimated by visual observation and physical inspection by“poking” the disks with a fine spatula. The porous membrane which hadbeen cut out while making the disks remained attached to the TET-FG andwas used to help position the disks during the evaluation of theirstructural integrity. Pictures of top and lateral views of the diskswere also taken and were used in the evaluation.

The structural integrity of FG and TET-FG were measured under bothsterile and non-sterile conditions. For the non-sterile experiments, thePBS and saliva were stored frozen until analyzed. For the sterileexperiments, the same procedure was used except that the entire processwas run under sterile conditions. The sterility of the system was testedby incubating 0.2 ml of sample and 2 ml of broth at 37° C. and theturbidity of the broth was monitored for 48 hours. Lack of turbidityindicated sterility of the system. The stability of the CIP-, AMO-, andMET-FG were studied as above but under non-sterile conditions only.

C. In Vitro Antimicrobial Activity of AB Released from AB-FG

The antimicrobial activity of the AB released from the AB-FG wasestimated by measuring the diameters of the zones of inhibitiongenerated by the eluate from the 6 mm diameter disks from the dailycollected PBS or saliva surrounding the AB-FG. The eluates fromunsupplemented FG served as controls. AB solutions of knownconcentration were used as standards. E. coli cultured on agar plateswere used to measure the AB activity of the released TET, CIP and MET.To make the culture plates, 100 μl of the bacterial cell suspension,containing approximately 10⁸ cells/ml, was mixed with 3 ml of top agarat 50° C. and immediately poured onto the plate hard, bottom agar tomake a uniform layer of cells. The plates were incubated at 37° C. for18 hours.

Results

A. TET

1. TET Release Data

The release of TET from TET-FG disks into the surrounding PBS in the“static” experiments was measured spectrophotometrically by determiningthe TET concentration achieved in the 2 ml of PBS which was replaceddaily. The TET concentrations which were obtained for different amountsof TET that had been incorporated into TET-FG are shown in FIG. 23. AtTET concentrations in the TET-FG of less than 50 mg/ml, the release ofTET was completed in five days or less. However, the release of TET fromTET-FG disks which contained TET concentrations of 100 and 200 mg/mloccurred for approximately two weeks, and more than three weeks,respectively. The structural integrity of the TET-FG disks was preservedfor three to five weeks. These results demonstrated that the TET releasewas independent of the FG degradation and that the rate of TET releasedepended on the amount of TET which remained in the TET-FG disks.

The spectrophotometric data which were collected in the continuousexchange experiment are shown in FIG. 24. These data indicate that acontinuous TET release from a TET-FG disk which originally contained aTET concentration of 100 mg/ml FG occurred over a two week period. TheFG disk retained its structural integrity during this two week period,infra. The TET release data obtained in the continuous mode experimentalso indicated that the rate of TET release opportunity depended on theconcentration of TET which remained in the TET-FG disk.

While not wishing to be bound by theory, it is believed that the initialhigh TET concentrations observed in these experiments were probably aconsequence of the diffusion of TET from at or near the disk's surface.That is, as the TET “trapped” at these locations was exhausted, the rateof solubilization and/or diffusion decreased in a fashion that was mostprobably determined by the TET concentration gradient and by the shapeor configuration of the FG.

Temperature and FG protein concentration also played a role indetermining the TET diffusion rate from the TET-FG disks (see Examples13 and 14), but these two parameters were kept constant in theseexperiments.

The release of TET into saliva from TET-FG containing 50 and 100 mg/mlof TET was measured in static experiments by determining the TETconcentration in 0.75 ml of saliva that was replaced daily. Theseresults (FIG. 25) are similar to those obtained in PBS except that theconcentration of TET was higher, most probably reflecting the smallervolume of saliva which was used to collect the released TET. Inaddition, the presence of TET in the FG matrix again unexpectedlyprolonged the structural integrity of the TET-FG matrices for at least15 days compared to that for the control FG disks which had begun todecay by 9 days and were almost completely decayed by 15 days (FIG. 26).

2. TET Antimicrobial Data

The antimicrobial effects on E. coli growth of several TETconcentrations in PBS are shown in FIG. 27. The lowest TET concentrationdetectable by this method was approximately 5 μg/ml. These resultsclearly indicate that the released TET has antimicrobial activity. TheseTET data corroborate those obtained by spectrophotometry and indicatethat the amount of TET incorporated into the FG determines the TETconcentration in the solution surrounding the TET-FG. These data alsodemonstrate that the amount of TET in the FG can be tailored to maintainthe desired TET concentration in the medium surrounding the TET-FG at orabove the minimum desired TET concentration.

3. TET-FG Matrix Longevity

The longevity of control FG and AB-FG disks was evaluated by visualassessment of the disks. The porous membrane, cut during the making ofthe disks, remained attached to the FG and helped to position the disksduring their integrity evaluation. Top views of disks containing no TET(controls), and 50 or 100 mg of TET per ml of FG are shown in FIG. 26 atdays 0, 9 and 15. This figure shows typical results, namely, the FGcontrol disks were degraded within two weeks whereas the TET-FG disksremained intact, or nearly so, for 15 days. In additional experimentsTET-FG disks remained intact or nearly so for at least five weeks (datenot shown). No significant change in the FG longevity was observedbetween sterile and non-sterile TET release experiments.

B. CIP, AMO and MET Data

1. CIP, AMO and MET Release Data

The antibiotic released from CIP-, AMO- and MET-FG is shown in FIG. 28.CIP was released at an apparent constant rate for approximately 4 weeksand then the rate decreased gradually for approximately one more week.The release of AMO and MET was complete within 3 days.

2. CIP and MET Antibacterial Activity

The antimicrobial activity of released CIP and MET (data not shown)parallels the profiles determined spectrophotometrically for identicalAB-FG disks.

3. Supplemented—FG Matrix Longevity

The results for CIP-FG were similar to those for TET-FG. The results forAMO- and MET-FG were similar to those obtained for the FG control. Nosignificant change in the FG longevity was observed between sterile andnon-sterile experiments.

Discussion

The results demonstrated that poorly water soluble forms of CIP and TETprovide a combination of factors that increase significantly the maximumAB load, release period and longevity of the FG matrix into which theyare mixed. Alternatively, the FG disks can be stabilized by immersingthem in solutions of AB such as TET or CIP.

The results also clearly showed that the AB delivered by AB-FG preservedits antimicrobial activity as demonstrated by the inhibition of E. coligrowth. These results demonstrated that TET and CIP supplementation ofFG and other TS can overcome the degradation of FG as a limiting factorin drug delivery therefrom. That is these ABs stabilized the FG so thattheir release periods and the released AB concentrations can becontrolled using AB concentrations in the FG. Using these procedures TETand CIP can be loaded into FG and their release can be controlled for aperiod of days or weeks at effective antimicrobial concentrations.

The TET- and CIP-induced FG stabilization can be exploited forcontrolling the total release time not only for these ABs, but also forother drugs or “supplements” added to FG whose release rate and/or totalrelease duration depends on the integrity of the FG matrix.

These results have clinical applications in periodontal and otherconditions where FG can serve as a localized drug delivery system. TheTET- or CIP-induced FG stabilization can be exploited for controllingthe total release time of TET, CIP and other drugs or supplements whichhave been added to the TET-FG or CIP-FG matrices.

EXAMPLE 15 Effect of Temperature on the TET Release Rate fromTET-Supplemented FG

FG was supplemented with 50 mg/ml of TET free base and was shaped as6×2.5 mm disks for this study. The protein concentration of FG wasadjusted to 60 mg/ml. The disks were placed in 2 ml of PBS, pH 7.3 andwere allowed to stand at 4, 23 and 37° C. To wash the disks, the PBS wasreplaced every 10 minutes, 6 times, with 2 ml of fresh PBS. Thereafterthe PBS was replaced every hour for 4 hours. The TET concentrations inthe collected samples were determined spectrophotometrically against astandard curve as before.

The results demonstrated that the rate of TET release was proportionalto the temperature (FIG. 29).

EXAMPLE 16 Effect of FG Protein Concentration on the TET Release Ratefrom TET-Supplemented FG

FG supplemented with 1 mg/ml of TET HCl solution was prepared and wasshaped as 6×2.5 mm disks for this study. The protein concentration ofthe FG was adjusted to 60, 30 and 15 mg/ml. Each disk was placed in 3 mlof distilled water. The water was replaced with the same volume of waterevery 10 minutes for a total of one hour. The TET concentration in thecollected samples was determined spectrophotometrically against astandard curve as before.

The data (FIG. 30) show that the TET release rate was highest from theFG with the lowest total protein concentration and vice versa. That is,the TET release rate was inversely proportional to the FG proteinconcentration.

EXAMPLE 17 In Vivo Antimicrobial Activity of AB Released fromAB-Supplemented FG

To test the antimicrobial activity of TET and CIP released from TET- andCIP-FG, the capacity of these AB-supplemented FGs to protect mice frominduced peritonitis was evaluated. Experimentally, at day 1, each one of5 animals per group were injected intraperitoneally with 0.5 ml PBS(Group-I), FG (Group-II), TET-FG (Group-III) or CIP-FG (Group IV). FGand AB-FG was administered using a Hamaedics dispenser containing 0.25ml of TFC at 120 mg/ml and 0.25 ml of human thrombin at 250 U/ml. In thecase of TET- and CIP-FG, the thrombin solution contained 50 mg of therespective AB. At day 2, all the animals were injected intraperitoneallywith 2×10⁸ (Experiment 1) or 4×10⁸ (Experiment 2) colony forming units(cfu) of S. aureus 202A. Results: (Experiment 1, Experiment 2. Animalssurviving at 48 hours after infection): Group I, 0 and 1 survivors;Group II, 3 and 1; Group III, 3 and 5; and Group IV, 5 and 4 survivors.Most survivors lived through the duration of the experiment (2 weeks)but some died or were intentionally killed because they were sick.

These data demonstrated that TET-FG and CIP-FG protected mice from deathcaused by S. aureaus 202A for at least 48 hours after the administrationof the AB-supplemented FG.

EXAMPLE 18 Therapeutic Applications of Supplemented Fibrin SealantCompositions

The development of ultrathin microfiberoptic endoscopes has offered thelaryngologist unique access to the limited spaces of the temporal boneand skull base. While diagnostic middle ear endoscopy is well documented(Edelstein, D. R. et al., Am. J. Oto. 15:50–55 (1994); Poe, D. S. etal., Laryngoscope 102:993–996 (1992); Poe, D. S. et al., Am. J. Oto.13:529–533 (1992); Balkany & Fradis, Am. J. Oto. 12:46–48 (1991)),therapeutic microendoscopy offers the exciting advantages to the patientof minimal invasiveness, reduced patient morbidity and lower hospitalcost. Microendoscopes of constantly shrinking diameters yield images ofgood quality and resolution. Coupled to a laser and fibrin sealantapplicator, several new surgical applications in the middle ear andskull base are now feasible. Potential therapeutic applications werederived from the fibrin sealant's mechanical properties in soft tissuerepair and use as a sustained delivery vehicle for pharmaceuticals andbiologic growth factors. Possibilities include ototopical aminoglycosidetherapy, using for example gentamycin for the treatment of Meniere'sdisease, transeustachian CSF leak prophylaxis and tympanic membranerepair.

Preliminary antibiotic “release profiles” were obtained using pooledfibrin sealant (American Red Cross, Rockville, Md.), and eitheramoxicillin and metronidazole as “water soluble” agents, or tetracyclineand ciprofloxacin in the “low solubility” category. For this procedure,four human head specimens were preserved and underwent latex vascularinjection using the fresh tissue cadaver protocol actively in progressin the Naval Medical Center San Diego, San Diego, Calif. (The freshtissue cadaver protocol is advantageous in preserving the specimenswithout loss of “fresh tissue” qualities.)

Both fiberoptic and rigid systems were used as provided by XomedCorporation (Jacksonville, Fla.). The Alphascope 8 model was a flexiblemicrofiberoptic endoscope with an outside diameter of 0.8 mm and a 115degree flexible tip which provides a field of view of 65° with 1.5–15 mmdepth of observation. The fiberoptic cable was composed of 3,000 pixelsand provides 10 cm of insertion length. The Alphascope 12A model was arigid endoscope with an outside diameter of 1.2 mm and an obliquelyangled shaft of 25° and tip of 45° which provided a field of view of 65°with 2–20 mm depth of observation. The fiberoptic cable was composed of6,000 pixels and provided 8 cm of insertion length. A 0.28 mm KTP laser(Laserscope, Palo Alto, Calif.) was used for all laser applications.

Limited-sink conditions were created using 6×3 mm fibrin sealant discsmixed with a set concentration of antibiotic. Concentrations in theeluate were measured on a daily basis (μg/ml) and evaluated over time todevelop the “release profile” in vitro.

A duo-flow catheter was designed specifically to facilitate endoscopicapplication of fibrin sealant, having a 0.75 mm inner cannula with a 1.5mm outer cannula. The 1.5 mm outer diameter allowed coupling to amicrofiberoptic endoscope for access to the middle ear space, eustachiantube and cranial cavity. A “coaxial,” recessed tip allowed continuoustissue sealant application under visual guidance without clotting of thedelivery ports.

Microendoscopic and Laser Techniques

Initial procedures were performed on human temporal bone specimens todocument the feasibility of microendoscopic work within the middle earand temporal bone. Both transtympanic as well as transeustachian tuberoutes were used to access the middle ear. All surgery in the posteriorcranial fossa was performed through “keyhole” incisions in the posteriorfossa dura through a suboccipital approach. Procedures utilized standardotologic equipment.

Coupled with the KTP laser, surgical manipulation was safely achievedaround the oval window, to include lysis of adhesions and stapedotomy.Through a “keyhole” retrosigmoid approach, the flexible endoscope wasintroduced into the posterior cranial fossa with ready identification ofthe 7–8 nerve complex. When a comfortable level of technical competencewas reached, the KTP laser was successfully employed for vestibularnerve section in 6 cadaver specimens without structural damage toneighboring neurovascular structures. Although difficulty wasencountered in gauging the depth of vaporization in the first twospecimens with damage apparent to the anteriorly located facial nerve,the problem was resolved with refinement of the technique and a changein the laser angle. The duo-flow catheter was attached to the endoscopewhen using the KTP laser to suction laser plume.

Fibrin Sealant Delivery

Coupled to a microfiberoptic endoscope, the Duo-Flow catheter (HemaedicsCorp., Malibu, Calif.) was used to deliver antimicrobialcomposition-supplemented fibrin sealant under direct view to theeustachian tube and middle ear space. Several routes of delivery wereused including transtympanic, transeustachian tube and transmastoidthrough the facial recess. Successful “sealing” of the middle earcavity, eustachian tube and mastoid cavities was achieved with eachmethod of delivery. Fibrin sealant was noted to persist in these“static” specimens for over one week following application.

Tetracycline release profiles from the fibrin sealant disks showed aprolonged decay pattern in excess of three weeks. Concentrations abovetherapeutic Minimum Inhibitory Concentrations (MICs) remained for up to42 days. Fibrinogen concentrations ranging from 20–76 mg/ml had littleeffect on the release profile of ciprofloxacin.

This demonstration of a sustained-release capacity of fibrin sealantdemonstrated the great potential of the supplemented fibrin sealantcomposition as a therapeutic delivery system. On the antimicrobiallevel, topical application of fibrin sealant allows long-term deliveryof antibiotic doses at many times the current minimal inhibitoryconcentration, often avoiding side effects observed in a systemictherapy. In particular, when coupled with the laser, microendoscopicsurgery using a fibrin sealant localized-release “bioreservoir” offersgreat potential in the treatment of a broad spectrum of otolaryngicdisorders ranging from ototopical amino-glycoside treatment of Meniere'sdisease to laser nerve section and topical antimicrobial therapy ofacute and chronic sinusitis and otitis.

EXAMPLE 19 Sustained Release of Antimicrobial Compositions from FibrinSealant

Fibrin sealant (FS) disks were made by the enzymatic conversion offibrinogen to fibrin by thrombin, and subsequently cross-linked byFactor XIII. Briefly, 100 mg of human Topical Fibrinogen Complex (TFC,American Red Cross, Rockville, Md.), containing 76% fibrinogen andFactor XIII, was combined with 10 mg human thrombin (American Red Cross,Rockville, Md.) and 0.9 ml 40 mM calcium chloride solution. Thecrosslinking fibrin clot was quickly placed into a 20×10×3 mm mold andpressed to form a slab. FS disks were then punched from the slab using a6 mm biopsy punch. Following the same procedure, antibiotics were mixedwith the lyophilized TFC and thrombin prior to hydration to formantibiotic-impregnated FS (AB-FS) disks. Tetracycline free-base,ampicillin free-acid and ciprofloxicin hydrochloride (Sigma ChemicalCo., St. Louis, Mo.) were added separately as 345 mg to the TFC andthrombin prior to calcium chloride addition (final fibrin concentrationwas 76 mg/ml; final antibiotic concentration was 50 mg/disk).

Antibiotic release was measured in vitro under two extreme conditions,“limited sink” and “infinite sink.” Under limited sink conditions, FSand AB-FS disks were placed individually into wells of a 24-well tissueculture plate with two ml of phosphate buffered saline (PBS, pH 7.4).Tissue culture plates were left at 37° C. without agitation. The totalvolume of PBS was exchanged daily and the eluates evaluated forantibiotic content. Under infinite sink conditions, FS and AB-FS diskswere placed individually into 50 ml conical centrifuge tubes with 45 mlPBS and agitated by inversion (20 times/min). All tubes were maintainedat 37° C. The total volume of PBS was exchanged daily and the eluatesevaluated for antibiotic content.

Antibiotic concentrations were calculated from linear standard curves ofoptical density versus concentration (0–200 ug/ml). Tetracycline sampleswere evaluated spectrophotometrically at 340 nm. Ampicillin was measuredby first reacting 0.1 ml of the eluate sample with 2.9 ml BCA reagent(Pierce Chemical Co., Rockford, Ill.) for 30 min at 37° C. The resultingcolored product was measured at 560 nm. Ciprofloxicin samples wereevaluated directly at 340 nm.

To evaluate antibiotic release in vivo, tetracycline (TET)-supplementedFS disks were implanted into mice at two different locations. MaleBALB/c mice (20–25 g) were anesthetized for the subcutaneous (s.c.) orintraperitoneal (i.p.) implantation of disks. Incision sites were closedwith resorbable sutures and stainless steel clips. Disks were removed at2, 7, 14, 21 or 28 days post implantation and enzymatically digestedwith 0.1% trypsin/0.4 mM EDTA at 37° C. for 4–7 days. TET concentrationsof the lysates were measured as above to determine the mass of TETremaining in disks after in vivo incubation.

To assess the bioavailability of the antibiotic in TET-FS disks, TET-FSdisks were placed into test tubes containing a log phase culture of S.aureus (1×10⁷ CFU/ml). Cultures with FS disks containing no antibioticsserved as controls. All cultures were incubated at 37° C. for 10 hr.Bacterial samples (0.1 ml) were serially diluted and plated ontonutrient agar to determine the viable bacterial count during theincubation with the disks. An unmanipulated culture was also monitoredfor comparison.

The elution profiles for the three antibiotics evaluated under limitedsink conditions are presented in FIG. 31A. After an initial burst ofantibiotic release, the freely water soluble ampicillin elutedcompletely from the supplemented FS matrix within 7 days. This contraststhe elution profile of tetracycline free-base which demonstrated aslowly decreasing, steady release over 42 days. Tetracycline elution atday 42 was a sustained, anti-microbially effective amount, 0.03–0.04mg/ml. The release kinetics for ciprofloxicin parallelled those oftetracycline; although, data were only collected for 14 days. Theelution profile for infinite sink conditions demonstrated an enhancedrelease of antibiotics during the first 7 days for all three antibioticscompared with limited sink conditions. Otherwise the elution profilesparalleled those observed for the limited sink conditions.

Release of tetracycline in vivo was measured by calculating theantibiotic remaining in AB-FS disks after 2, 7, 14, 21 or 28 days of invivo implantion. The data are presented in FIG. 31B (combined with invitro data) and show that the elution profile for TET-FS disks parallelsthe elution profile of the limited sink model in vitro. After 14 days invivo, TET-FS disks still contained 50% of the starting concentrationwith no difference observed between the two sites (˜20% i.p. at day 28).These data demonstrate that both the s.c. and the i.p. sites facilitatedthe long-term delivery of TET from the TET-FS disks, and that the invitro experiments were highly predictive of the demonstrated in vivotherapeutic effect.

Antibacterial activity was determined by the ability of TET-FS disks toinhibit growth of a S. aureus culture in vitro (FIG. 31C). TET-FS diskssignificantly inhibited bacterial growth in the 10 hr of study ascompared with FS disks alone. Release of tetracycline and ciprofloxicinfrom FS disks was long term in both in vitro models demonstrating thecorrelation between the long term delivery of antibiotics andsolubility. Antibiotics of relatively lower solubility were consistentlyreleased over longer time periods than highly soluble preparations. Thedelivery kinetics in vivo resemble those of the limited sink modelsuggesting a limited flow of body fluids at the s.c. and i.p. sites ofdelivery. Supplemented FS disks were shown to provide long-term deliveryof concentrations of antibiotic sufficient to effectively inhibitbacterial growth, demonstrating that FS is an ideal, biocompatible,resorbable delivery system capable of releasing efficacious localizeddoses of antibiotic over an extended period of time.

EXAMPLE 20 Long Term Site-Directed Delivery ofCytotoxic/Antiproliferative Drugs from FG

The fibrinogen was solubilized with sterile water or, for one group withwater saturated with 5-FU at a concentration of 17 mg/ml. Thrombinsolutions were made with sterile water, and then were diluted in 40 mMCaCl₂ to a concentration of 15 U/ml, or Thrombin was dissolved in 40 mMCaCl₂ saturated with 5-FU in a concentration of 17 mg/ml.

Control FG clots did not contain 5-FU and were produced by mixing 200 μlof TFC solution (at 60 mg/ml) with 200 μl of Thrombin solution (at 15U/ml) and allowing 20 minutes to polymerize. These clots were made in 12by 75 mm test tubes and then were placed in 10 mls of 0.05 M Histidine,0.15 M NaCl, pH 7.3 (Buffer).

FG clots containing saturated levels of liquid 5-FU were produced bymixing 200 μl of TFC (60 mg/ml+17 mg/ml 5-FU) with 200 μl Thrombinsolution (15 U/ml+17 mg/ml 5-FU) and allowing 20 minutes for the clotsto fully polymerize. The addition of saturated levels of 5-FU in boththe TFC and Thrombin solutions somewhat altered clot formation producinga clot that was translucent, as compared to the control FG clots whichwere quite opaque. The clots that were formed were physically the sameas those made with FG alone except in color. Clots were formed in 12 by75 mm test tubes and then placed in 10 ml of buffer.

A second group of FG clots were made that contained an amount of solidanhydrous 5-FU equal to the amount included in clots formed withsaturated solutions of 5-FU. These clots were formed by the addition of7 mg of solid anhydrous 5-FU to 200 μl of TFC (60 mg/ml) and 200 μl ofThrombin (15 U/ml). Seven mg of 5-FU was placed in a 12 by 75 mm testtube. Two hundred μl of TFC was then added followed by 200 μl ofThrombin. The 3 components were then mixed by pipetting back and forthuntil a homogenous mixture was observed and further mixing was inhibiteddue to the clotting reaction. Clots were then placed in 10 ml ofhistidine buffer.

The final group contained 50 mg of solid anhydrous 5-FU per clot. Due tothe increased mass of 5-FU (50 mg instead of 7 mg) the previously usedmethod did not work. Instead of producing a homogenous clot, a clot wasformed with the majority of the 5-FU having settled to the bottom of thetest tube. To avoid this problem the bottom of the test tube was firstcoated with 100 μl of TFC (60 mg/ml) and 100 μl of Thrombin (15 U/ml).This formed a clot which covered the concave bottom of the test tube.Next, 50 mg of solid anhydrous 5-FU was added to the surface of the 200μl clot. Following this, 100 μl of TFC was added along with 100 μl ofThrombin. The two solutions were mixed using an automatic pipettor untilthe protein started to gel. When this occurred, the pipetting was endedand the clot was allowed to polymerize for 20 minutes. The final productwas a clot that contained a dense core of approximately 50 mg of 5-FU.As with the other clots, these were then placed in 10 mls of buffer. Thefinal total protein concentration of the FG in all groups was 30 mg/ml.

Each group contained 10 replicates. Each duplicate was incubated at 37°C. in 10 mls of buffer. Buffer was exchanged for 10 mls of freshsolution at 5, 10, 22, 33, 52, 75 and 114 hours. Aliquots of the eluatebuffer were then examined in a spectrophotometer at a wavelength settingof 260 nm. Previous experiments had demonstrated that 5-FU absorbedstrongly at this wavelength, while eluates from control FG clots didnot.

The results are shown in FIG. 32. Control clots containing no 5-FU gaveno significant readings. Clots made with 7 mg of 5-FU either in the formof saturated solutions of 5-FU or an equivalent amount of solidanhydrous 5-FU completed their delivery of 5-FU between 5 to 10 hours,while the clots containing 50 mg of solid anhydrous 5-FU continued todeliver 5-FU for at least 75 hours. Peak levels in all cases occurred atthe 5 hour time point.

While not wishing to be bound by theory, it is believed that theduration of 5-FU delivery appeared to be a function of the mass of 5-FUloaded into the gel. As a result, the amount of 5-FU deliverable fromthe clots containing 5-FU in solution was limited by the solubility ofthe drug. Thus the inclusion of amounts of solid anhydrous form equal tothe amount present in the clots formed from liquid saturated with 5-FUresulted in nearly identical delivery kinetics, while the inclusion ofgreater amounts of 5-FU in the solid form than were possible using theliquid form, resulted in a tripling of the total duration of delivery,and typically a 10-fold increase in the duration of delivery of a givenconcentration of the drug. It would be expected that the inclusion ofstill greater amounts of the solid anhydrous 5-FU would also result ineven greater delivery times. In other experiments, it has been foundthat the amount of 5-FU included in the clots can be increased at least5-fold and probably higher, and that the 5-FU-FG mixture can also beformulated into an injectable form (data not shown). It would further beexpected that the use of an analog or other form of 5-FU that was lesssoluble in the surrounding aqueous medium than the anhydrous form,and/or had a slower dissolution rate, would result in a further increasein delivery times.

The result of this process is a sustainable delivery of theantiproliferative/cytotoxic drug 5-FU from fibrin clots for at least 10times longer than is possible using the drug in the aqueous form. Thistechnology (i.e., the use of a solid form of the drug, preferably onewith a low solubility and/or dissolution rate) should be generallyapplicable regardless of the matrix in which the drug particles aresuspended, or the drug itself.

EXAMPLE 21 Delivery of Taxol from Fibrin Sealant

Based upon the successful controlled delivery of 5-FU from asupplemented fibrin sealant matrix, protocols were developed to considerthe delivery of other chemotherapeutic compounds. Recently, paclitaxelor taxol has been recognized as a very promising agent for the treatmentof ovarian and breast cancers (Nicoletti et al., Ann. of Oncology 2:151(1993)). One problem with administering taxol, systemically is that itis highly insoluble in aqueous systems. This has necessitated the use ofa systemic delivery vehicle consisting of an oil and alcohol mixture(Rose, W., Anti-cancer Drugs 3:311 (1992)). Unfortunately, this systemicdelivery vehicle causes severe reactions in many patients, and currenttherapeutic applications call for pre-medication to minimize them(Weiss, et al., J. Clin. Oncol. 8:1263(1990); Arbuck et al., Seminars inOncol. 20:31(1993). The malignancies for which taxol is currently underclinical use are generally slow-growing, suggesting that an extendedexposure to taxol from supplemented fibrin sealant would be desirable.Additionally, since the lesion produced by these diseases is oftenaccessible clinically through percutaneous biopsy or laparoscopicprocedures, the prolonged delivery of effective local concentrations oftaxol from a fibrin sealant matrix appeared therapeutically feasible.

The kinetics of taxol delivery from fibrin sealant were initiallyevaluated, by incorporating taxol (0.26 mg), either as an anhydroussolid or dissolved in ethanol, into a 400 μl fibrin sealant composition.The resulting supplemented fibrin matrices were then placed in 2 mlhistidine buffer, and incubated at 37° C. The buffer was exchanged aftertwo days, and again ten days later. The relative concentration of taxolin the resulting eluates determined by measuring their ability toinhibit the growth of a human ovarian carcinoma cell (OVCAR) in vitro(MacPhee et al., In Current Trends in Surgical Tissue Adhesives:Proceedings of the First International Symposium on Surgical Adhesives,R. Saltz and D. Sierra, eds. Springer-Verlag).

Briefly, 1000 OVCAR cells in 100 μl of growth medium were plated intoeach well of a 96 well culture plate and incubated for 24 hours. A 100μl volume of various dilutions of the eluates was then placed into thewells (10 wells per dilution), and the plates incubated at 37° C. Afterfive days, the number of cells in each well was measured using the MTTassay (Rapaport et al., American Journal of Clinical Pathology 97:84(1992)). In this assay, the effect of an anti-proliferative agent isseen as a decrease in the number of cells in the final cultures, andconsequently, as a decrease in the amount of MTT that is converted intoa chromaphore. The resulting chromaphore is detected byspectrophotometry at 570 nm. The results of the experiment and thesource of each eluate is provided in FIG. 33. (p<0.001 relative to themedium control (Dunns test)).

The controls included an initial (cellular) activity control (IAC)showing the amount of substrate produced by the OVCAR cells at the timeof addition of the eluates, and the medium control, showing the maximumamount of substrate produced after 5 days in culture. The eluates fromunsupplemented fibrin sealant alone did not affect this growth.

The results obtained using taxol in solution in ethanol showed that thetaxol was completely delivered for up to 85 days. When taxol wasincorporated into the fibrin sealant in the solid anhydrous form, theOVCAR cells were significantly inhibited for up to 85 days. Subsequenteluates recovered after an additional 10 days in culture (day 12eluates) also significantly inhibited the growth of OVCAR cells equallywell at dilutions from 1:200 to 1:20,000. This indicated that when thefibrin matrix is supplemented with the solid form of taxol, delivery wassustained beyond the initial 2 day period, and that the amount of taxoldelivered in the period from day 2 to day 12 exceeded that which wasdelivered in the first 48 hours.

These experiments showed that long term delivery of taxol from asupplemented fibrin sealant composition can be accomplished by loading amass of drug that exceeds its solubility in the matrix volume. This waspossible both by incorporating the taxol in its solid form, as well asby dissolving it in ethanol prior to incorporation. This is because themolecular weight of ethanol is much lower than that of taxol. As aresult, ethanol will rapidly diffuse from the matrix leaving the highlywater insoluble taxol behind to precipitate into solid form within thematrix.

EXAMPLE 22 Fibroblast Chemotaxis in Response to Fibroblast GrowthFactor-Supplemented FG and Fibronectin

Dulbecco's Modified Eagle's Medium (DMEM) was purchased from SigmaChemical Co., St. Louis, Mo. Antibiotic-Antimycotic solution waspurchased from GIBCO (Grand Island, N.Y.). Recombinant fibroblast growthfactor-1 (FGF-1) and -4 (FGF-4) were a kind gift of Reginald Kidd,Plasma Derivatives Laboratory, American Red Cross, Rockville, Md., andGenetics Institute (Cambridge, Mass.), respectively. Recombinantfibroblast growth factor-2 (FGF-2, also known as basic FGF or bFGF) waspurchased from Upstate Biotechnology, Inc. (Lake Placid, N.Y.) Allplastic ware required for sterile propagation of cultures as well as thechemotaxis assays were purchased from Fisher Scientific (Newark, Del.).Millicell-PCF (12.0 μm) inserts were purchased from Millipore, Inc.(Bedford, Mass.). Heparin was obtained from the UpJohn Company(Kalamazoo, Mich.).

NIH/3T3 fibroblasts at passage 126 were purchased from the American TypeCulture Collection, Rockville, Md. Cultures from passages 129–133 wereused in the chemotaxis assays. Cultures were propagated in DMEMsupplemented with 10% Calf serum and approximately 1% antibioticantimycotic solution. Human dermal fibroblasts (HDFs) were purchasedfrom Clonetics, Inc. (San Diego, Calif.) at passage 2. Cultures frompassages 3–5 were used in the chemotaxis assays. Cultures werecultivated in DMEM supplemented with 20% FBS (Hyclone Laboratories,Inc., Logan, Utah) and approximately 1% antibiotic antimycotic solution(Gibco, Grand Island, N.Y.).

Cell Chemotaxis Assays

The procedure used to determine cellular chemotaxis was a combination oftwo known methodologies. A modification of Boyden's chamber was used asfollows: Millicell-PCF (Millipore, Inc., Bedford, Mass.) (12.0 μm) 12.0mm diameter inserts were placed in individual wells of 24 well plates tocreate the upper and lower chemotaxis chambers. Chemotaxis results werearrived at by performing checkerboard analysis for every combination ofcells and growth factors. Concentrations ranging from 0.1, 1, 10, 100ng/ml with/without added heparin (10 U/ml) were used for FGF-1, FGF-2(no heparin) and FGF-4 with all the cell types mentioned in thematerials section. Briefly, cultures were trypsinized and placed inDMEM+0.1% Bovine Serum Albumin (BSA) (Sigma Chemical Co., St. Louis,Mo.) for approximately one hour at 37° C. in a 5% CO₂ humidifiedchamber. Two to 2.5×10⁵ cells in 50 μl were added per insert to theupper chamber of the setup of the 24 well plates. Treatments were addedas mentioned above. The assay was kept at 37° C. in a 5% CO₂ humidifiedchamber for 4 hours. All combinations tested were performed intriplicate. At the end of 4 hours, the plates were removed from theincubator and the filters were stained following the protocol forstaining included with the Millicell-PCF inserts. Briefly, the fluidsurrounding the inside and outside of the Millicell-PCF inserts wasremoved. Three percent glutaraldehyde (Sigma Chemical Co., St. Louis,Mo.) was added to the outside and inside of the inserts forapproximately 20 minutes. Following removal of the 3.0% glutaraldehyde,0.5% Triton X-100 (E.M. Science, Chemy Hill, N.J.) was added for 5–7minutes. On removal of the 0.5% Triton X-100, Fisher's HematoxylinSolution Gill's Formulation (Fisher Scientific, Newark, Del.) No.1 wasadded for about 10 minutes. This solution was washed off in runningdistilled water for about 5 minutes. Using a cotton swab the upper sideof the filter was swabbed to remove cells which had not migrated.Filters were mounted lower side facing up on slides in Crystal Mount™(Biomeda, Inc., Foster City, Calif.) solution and 10 random fields werecounted per slide both visually at 400× and at 200× using an ImageAnalyzing System to automate the enumeration of the cells on theunderside of the filters.

Checkerboard Analysis

As required, checkerboard analysis was carried out to determine randommigration, and positive and negative chemotaxis. Growth factors wereadded to the upper and/or lower chambers to observe whether cellsmigrated towards the GF alone (chemotaxis), whether migration was randomirrespective of whether the growth factor was added to the upper orlower well (chemokinesis) or whether cell migration was against thechemotactic gradient (negative chemotaxis).

Cell Migration Assay to FGF Released from FG

Chemotaxis chambers and cells were utilized as described above. Fifty μlof 8 mg/ml Topical Fibrinogen Complex (TFC, American Red Cross,Rockville, Md.) was added to the bottom of 24 well plates. Forty μl oftest growth factor +/− heparin at a final concentration of 10 U/ml(FGF-1, FGF-4 with heparin, FGF-2 alone) was added to the TFC andthoroughly mixed. Ten μl of bovine Thrombin (Armour PharmaceuticalCompany, Kankakee, Ill.) was added and mixed thoroughly. The componentswere allowed to gel at room temperature for approximately 30 minutes.Total volume in the lower and upper chambers was made up to 0.5 ml eachwith DMEM+0.1% BSA. The concentration of the FGF's added to the TFC wasadjusted to produce the desired overall concentration as determined by:

${{Overall}\mspace{14mu}{FGF}\mspace{14mu}{Concentration}} = \frac{{mg}\mspace{14mu}{of}\mspace{14mu}{FGF}\mspace{14mu}{added}\mspace{14mu}{to}\mspace{14mu}{TFC}}{\begin{matrix}{{{Volume}\mspace{14mu}{of}\mspace{14mu}{liquid}\mspace{14mu}{in}\mspace{14mu}{upper}\mspace{14mu}{chamber}} +} \\{{{{Volume}\mspace{14mu}{of}\mspace{14mu}{FG}}\&}\mspace{14mu}{liquid}\mspace{14mu}{in}\mspace{14mu}{lower}\mspace{14mu}{chamber}}\end{matrix}}$The assay was performed at 37° C. in a 5% CO₂ humidified chamber forapproximately 24 hours. At the end of 24 hours, the filters wereremoved, fixed and stained and the number of cells on the underside ofthe filter was enumerated as described above.ResultsCapacity for Migration of Fibroblasts

The ability of NIH 3T3 fibroblasts to migrate towards various well knownchemotactic agents was determined to ensure that the cells used in thisassay retained this capacity. Fibronectin was the most effectivechemotactic agent tested for both NIH 3T3 and HDFs with maximalresponses occurring at 20 μg/ml (FIG. 34, Table 8). Thereafter,fibronectin at 20 μg/ml was used as the positive control for migration.

Chemotaxis of NIH 3T3 Fibroblasts Towards FGF-1

Maximum stimulation of migration of NIH 3T3 fibroblasts by FGF-1 wasobserved at 10 ng/ml in the presence of 10 U/ml of heparin (FIG. 35).Checkerboard analysis revealed that FGF-1 was chemotactic for NIH 3T3cells (Table 9).

Chemotaxis of NIH 3T3 Fibroblasts Towards FGF-2

Maximum stimulation of migration of NIH 3T3 fibroblasts by FGF-2 wasobserved at 1 ng/ml of FGF-2 (FIG. 36). Checkerboard analysis showedthat FGF-2 was chemotactic for NIH 3T3 cells (data not shown).

Chemotaxis of NIH 3T3 Fibroblasts Towards FGF-4

Maximum stimulation of migration of NIH 3T3 fibroblasts by FGF-4 wasobserved at 10 ng/ml (FIG. 37). Checkerboard analysis revealed thatFGF-4 was chemotactic for NIH 3T3 cells (data not shown).

Chemotaxis of HDFs Towards FGF-1

Maximum stimulation of migration of HDFs by FGF-1 was observed from 1 to10 ng/ml (FIG. 38). Checkerboard analysis showed that FGF-1 waschemotactic for HDFs (Table 10).

Chemotaxis of HDFs Towards FGF-2

Maximum stimulation of migration of HDFs by FGF-2 was observed at 10ng/ml (FIG. 39). Checkerboard analysis revealed that FGF-2 waschemotactic for HDFs (data not shown).

Chemotaxis of HDFs Towards FGF-4

Maximum stimulation of migration of HDFs by FGF-4 was observed at 10ng/ml (FIG. 40). Checkerboard analysis showed that FGF-4 was chemotacticfor HDFs (data not shown).

Human Dermal Fibroblast Migration to FGF-1, -2 and -4 Incorporated in FG

Maximal migratory response to FGF released from FG was elicited at anincorporated and total concentration of FGF-4 in FG of 1 ng/ml (FIG.41). Similar results were also found when FGF-1 and FGF-2 wereincorporated into the FG (data not shown) except that the concentrationof FGF-2 that elicited the peak chemotactic response was 0.01 mg/ml.

TABLE 8 Concentration of Fibronectin In Lower Concentration ofFibronectin In Upper Compartment Compartment 0 μg/ml 10 μg/ml 20 μg/ml50 μg/ml  0 μg/ml 48.53 +/− 4.695  62.3 +/− 3.269  69.6 +/− 12.25  62.0+/− 2.616 10 μg/ml  68.03 +/− 10.793 47.53 +/− 5.605 64.86 +/− 7.96174.66 +/− 3.946 20 μg/ml 90.53 +/− 5.203 88.73 +/− 4.152  56.9 +/− 3.289 76.23 +/− 1.8190 50 μg/ml 72.43 +/− 8.276  91.3 +/− 1.003 63.26 +/−3.835 57.46 +/− 2.287

TABLE 9 Concentration of FGF-1 In Lower Concentration of FGF-1 In UpperCompartment Compartment 0 ng/ml 1 ng/ml 5 ng/ml 10 ng/ml 0 ng/ml  32.1+/− 6.328 53.93 +/− 4.152 27.27 +/− 3.873 25.96 +/− 4.151 1 ng/ml 59.46+/− 6.89   36.9 +/− 5.728  22.1 +/− 9.232 35.86 +/− 2.074 5 ng/ml 64.867+/− 1.75  41.44 +/− 1.866 24.84 +/− 4.337  41.6 +/− 6.717 10 ng/ml 70.83 +/− 2.752 39.73 +/− 2.428 39.73 +/− 2.428 41.83 +/− 6.879

TABLE 10 Concentration of FGF-1 In Lower Concentration of FGF-1 In UpperCompartment Compartment 0 ng/ml 0.1 ng/ml 1 ng/ml 10 ng/ml 100 ng/ml   0ng/ml 1.96 +/− .602  48. +/− 1.965  1.3 +/− 0.351  4.6 +/− 2.424  2.4+/− 2.59 0.1 ng/ml 66.46 +/− 3.304  3.5 +/− 1.550  22.0 +/− 6.621  9.7+/− 7.758  11.6 +/− 8.609   1 ng/ml  90.0 +/− 5.776 27.7 +/− 8.10  52.6+/− 2.775  9.7 +/− 3.553 1.83 +/− 2.2   10 ng/ml  92.4 +/− 29.307  55.1+/− 7.151  44.2 +/− 11.844  16.2 +/− 4.781 21.2 +/− 6.42 100 ng/ml  65.4 +/− 22.055  53.7 +/− 7.3118  54.9 +/− 18.599 49.166 +/− 9.152 4.66 +/− 3.3 Discussion

The FGFs produced a profound chemotactic response in HDFs. For everychemotactic assay performed with HDFs, a very good distinction wasobtained between the negative control and the concentration of FGF whichelicited a maximal migratory response: 18, 12 and 10 fold in response toFGF-1, -2 and -4, respectively.

The stimulation of chemotaxis by growth factors was not as high for NIH3T3 cells as it was for HDFs, possibly due to the high passage number ofthe available stock cultures of the NIH 3T3 cells as compared to theHDFs.

FGF-1, FGF-2 and FGF-4 were found to be potent stimulators of fibroblastchemotaxis. Directed migration of fibroblasts by one or a combination ofthe above growth factors could result in fibroblast presence in the siteof injury, thereby leading to fibroplasia and the laying down ofcollagen and an extracellular matrix. Thus, aside from it's wellrecognized angiogenic properties, FGF's may have a role in woundhealing, acting either alone or in a combination with PDGF, IGF-I, TGF-βand/or other factors.

Previous studies into the use of FGF's to speed wound healing have notyielded significant results (Carter et al., 1988). This may be due to arequirement for the prolonged exposure of cells to the factors in vivofor a maximal response (Presta et al., Cell Regul. 2:719–726 (1991) andRusnati et al., J. Cell. Physiol. 154:152–161 (1993)). Unfortunately, itis difficult to deliver growth factors to wounds for such long timeperiods under conditions that would not interfere with the healingprocess.

The present invention of incorporating FGFs into FG allows for theprolonged exposure of cells to the FGFs and can be applied to a wound.The resulting fibrin coating mimics the natural response to tissueinjury, while delivering the growth factor directly to the wound site.In a previous study by the present inventors, FG which contained FGF-1was used to line artificial vascular grafts (Example 8, herein). Whenthese grafts were placed into the vessels of rabbits, the FGF-1 wasreleased for a period of up to 28 days. In further studies involvingcanine grafts, the effect of the incorporation of FGF-1 into the graftwalls was the total endothelialization of the artificial grafts withinthe same period (Greisler et al., Surgery 112:244–255 (1992)). Thus,this form of application elicits a profound biological effect in vivo.The fibroblasts are attracted towards FGF released from FG. Thisproperty will be useful in treating wounds with GF-supplemented TS.

EXAMPLE 23 Site-Directed Angiogenesis Using TS to Deliver AngiogenicSubstances

This embodiment permits the directed generation of new blood vessels ina controlled manner within the body. In this embodiment, the TS containsand delivers angiogenic substances, such as Fibroblast Growth Factor-1(FGF-1), in an amount such that its concentration which is released fromthe supplemented TS is effective to induce angiogenesis.

This embodiment is used in a controlled manner to revascularize bodyareas which have been deprived of an adequate blood supply such ascardiac, brain and muscle tissue, and the retina. This embodiment isused to restore or improve circulation to implanted organs orre-attached limbs. This embodiment can be used to generate a vascularnetwork or “vascular bed” for: the generation of artificial organs ororganoids, the delivery and/or localization of and/or nourishment ofcells used in gene therapy, or as a target of gene therapy, for thenourishment and/or localization of cells for tissue augmentation. Thisembodiment also precludes the necessity of implantation of a device orsubstance which may induce a foreign body or other excessiveinflammatory reaction which could compromise the blood vessel formationor the function of the underlying organ(s).

The invention consists of a formulation of fibrinogen, (suitable for theformation of fibrin) with or without fibronectin and/or collagen, intowhich is placed an appropriate concentration of an angiogenic substance,such as FGF-1. The fibrinogen may also contain stabilizers to protectagainst the proteolytic activity of Thrombin. In the case of FGF-1,heparin sulfate (1–1000 U/ml) may be used as the stabilizer in the rangeof concentration of from 1 ng/ml to 1 mg/ml. Alternatively theangiogenic substance is contained, in an appropriate concentration, inthe thrombin, calcium, or water components. This formation is then mixedwith thrombin and rapidly applied within the body in a line connectingthe desired sites, or to a single site. The fibrinogen-thrombin mix thenpolymerizes to form FG. The FGF-1, or other angiogenic substance,remains trapped in the FG matrix, either as a free form or bound to thestabilizer or another component of the mixture. In one embodiment, theconcentration of the FGF-1 in the TS should be from 0.1 ng/ml to 1mg/ml, more preferably from 1 ng/ml to 100 μg/ml, most preferably from100 ng/ml to 10 μg/ml. The FGF-1, or other angiogenic substance, willinduce blood vessel formation within the body of the deposited FG. TheFG will be naturally biodegraded leaving the intact blood vessel(s).

EXAMPLE 24 Site-Directed Cartilage Induction

This embodiment permits the controlled generation of new cartilage aswell as the guided regeneration of damaged cartilage within the body. Inthis embodiment the TS contains and delivers a cartilage promotingfactor(s), such as cartilage-inducing factors-A and/or -B (CIF-A andCIF-B, respectively, which are also known as TGF-B₁ and TGF-B₂,respectively) and/or another, factor(s) such as Osteoid-Inducing Factor(OIF) in an amount such that the concentration of the inducing factor(s)which is released from the supplemented TS is effective to inducecartilage formation. In one embodiment the concentration of the inducingfactors should be 0.1 ng/ml to 1 mg/ml, more preferably from 1 ng/ml to500 ng/ml, most preferably from 100 to 250 ng/ml. This embodiment mayalso contain drugs, such as antibiotics, and other growth factors, suchas EGF, PDGF, and bFGF in the TS. The cartilage inducing substance iscontained in an appropriate concentration in the fibrinogen or thrombinor calcium or water component(s) which are used to prepare the TS.

The supplemented TS can either be pre-shaped to the desired finalcartilage form prior to implantation or it can be implanted into thebody of the recipient in the liquid form as the TS is mixed andpolymerizes. The resulting form may then be sculpted as desired toproduce the required shape of cartilage needed. The Cartilage InducingTS (CI-TS) mixture can also be used to precoat a conventional implant,with the result being a conventional implant with a coating of livingcartilage.

Using any of the techniques described above, the CI-TS is then implantedinto the body of the recipient. This implantation can be heterotopic ororthotopic. After an appropriate interval, the CI-TS is be replaced byliving cartilage with the form of the original CI-TS implant.

Such implants can be used to replace damaged or lost cartilage, or toimprove the tissue integration and/or function of an artificial implant.Examples of such uses include the replacement or reconstruction of nasalor ear tissue, the generation of a functional joint surface on a boneimplant grown in vivo, or the generation of a similar surface on anartificial implant. The repair of cartilage damaged by disease, such asrheumatoid arthritis, can also be accomplished using the CI-TS toproduce a new and smooth cartilage surface to the arthus. Implantsintended for space filling applications in Plastic/Reconstructivesurgery can also be either formed from CI-TS, or coated with CI-TS toenhance tissue integration and reduce foreign body reactions.

Since current technology does not permit the guided regeneration ofcartilage, this invention is an advancement because it permits thegeneration of cartilaginous tissue which is required to fully mimic thebody's natural make-up. This results in improved joint repair,artificial joints and other implants, both for orthopedic and otherapplications.

For example, this embodiment can be used: to produce improved orthopedicimplants or improved plastic/reconstructive implants: for joint repairfor traumatic, congenital or pathologically damaged or dysfunctionalcartilage; to produce coatings of pacemaker implants and wires toincrease their tissue integration and to reduce foreign body reactions.Similar coatings could also be applied to any implantable device for thesimilar purposes.

EXAMPLE 25 Supplemented TS as a Surface Coating for Biomaterials

This embodiment uses supplemented TS as a coating for the surfaces oforthopedic devices and other biomaterials which are to be implanted intoan animal's body. Examples of these devices are urinary catheters,intravascular catheters, sutures, vascular prostheses, intraocularlenses, contact lenses, heart valves, shoulder/elbow/hip/kneereplacement devices, total artificial hearts, etc. Unfortunately, thesebiomaterials may become sites for bacterial adhesion and colonization,which eventually may lead to clinical infection that will endanger thelife of the animal. To minimize this problem, the biomaterial is coatedwith a supplemented TS.

In this embodiment the TS can be supplemented with: a growth factor(s);a drug(s), such as an antibiotic; BMP; and/or cultured cells, etc.Examples of antibiotics that may be incorporated into the TS include,but are not limited to: the penicillins; cephalosporins; tetracyclines;chloramphenicols; metronidazoles; and aminoglycosides. Examples ofgrowth factors which may be incorporated into the TS include but are notlimited to FGF, PDGF, TGF-β. Examples of BMPs which may be incorporatedinto the TS include, but are not limited to, BMP 1 through 8. DBM canalso be added to the TS. Examples of cultured cells which may beincorporated into the TS include, but are not limited to, endothelialcells, osteoblasts, fibroblasts, etc.

The supplement(s) may be contained in either the thrombin, fibrinogen,calcium or water component(s). The concentration of the supplement inthe TS is adequate such that it will be effective for its intendedpurpose, e.g., an antibiotic will inhibit the growth of microbes on thebiomaterial, a growth factor will induce the growth of the desired celltype(s) in the TS and/or on the surface of the biomaterial.

This invention is an improvement for existing biomaterial products,which include titanium and titanium alloy devices (such as fixationplates, shoulder/elbow/hip/knee replacement devices, osseointegrateddental implants, etc), solid silicone products (such as Silastic nasalimplants, liquid and/or gel silicone products (such as breast implantsand testicular implants), and natural or synthetic polymers used asconventional materials in healing a wound site, which may have variousforms, such as monofilaments, fibrous assemblies (such as cotton, paper,nonwoven fabrics), films, sponges, bags, etc.

FG is produced from 3 components: fibrinogen (for example as TFC); andthrombin, both of which may be in the lyophilized form; as well ascalcium. The lyophilized fibrinogen is reconstituted with sterile water,while the thrombin component is reconstituted with calcium chloridesolution. A supplement may be added to any of the three components priorto mixing. Appropriate volumes of the fibrinogen and thrombin containingcalcium are mixed to produce the FG. The FG is then applied to thebiomaterial's surface as a coating thereof as, for example, by spraying,painting, etc. Alternatively, the implant is dipped in the FG while itis still liquid. A supplement may also be added to the FG before orafter it has been coated on a biomaterial surface. For instance, aFG-coated implant is soaked in an antibiotic solution for a specifiedperiod of time so that the antibiotic will diffuse into the TS. Anotherexample is coating a device with TS after which cultured cells areseeded onto the fibrin coating. Coating the surface of biomaterials,which will be implanted into an animal, with supplemented TS will serveseveral purposes, including: the inhibition of bacterial adhesion to thebiomaterial; the inhibition of growth of bacteria adhered to thebiomaterial; local immune stimulation and/or normalization; thepromotion of would healing; and the promotion of engraftment of thebiomaterial to the surrounding tissue.

EXAMPLE 26 Self-Contained, TS Wound Dressing

This embodiment is a self-contained TS wound dressing, or bandage, whichcontains both the thrombin and fibrinogen components of the FG. Thecalcium is contained in either the thrombin and/or the fibrinogencomponent(s). Either or both of the thrombin or fibrinogen componentscan be, but does not have to be, supplemented with a growth factor(s),such as a FGF or bFGF, or a drug(s) such as, an analgesic, antibiotic orother drug(s), which can inhibit infection, promote wound healing and/orinhibit scar formation. The supplement(s) is at a concentration in theTS such that it will be effective for its intended purpose, e.g., anantibiotic will inhibit the growth of microbes, an analgesic willrelieve pain.

The thrombin and fibrinogen are separated from each other by animpermeable membrane, and the pair are covered with another suchmembrane. The thrombin and fibrinogen are contained in a quickevaporating gel (e.g., methylcellulose/alcohol/water). The bandage maybe coated on the surface that is in contact with the gel in order toinsure that the gel pad remains in place during use. (See FIG. 42).

In operation, the membrane separating the two components is removed,allowing the two components to mix. The outer membrane is then removedand the bandage is applied to the wound site. The action of the thrombinand other components of the fibrinogen preparation cause the conversionof the fibrinogen to fibrin, just as they do with any application of FS.This results in a natural inhibition of blood and fluid loss from thewound, and the establishment of a natural barrier to infection.

In a similar embodiment, the thrombin component and the plastic filmseparating the Thrombin gel and the Fibrinogen gel may be omitted. Thecalcium that was previously in the Thrombin gel may or may not beincluded in the Fibrinogen gel as desired. In operation, the outerimpervious plastic film is removed and the bandage applied, aspreviously described, directly to the wound site. The Thrombin andcalcium naturally present at the wound site then induce the conversionof fibrinogen to fibrin and inhibit blood and fluid loss from the woundas above. This embodiment has the advantage of being simpler, cheaper,and easier to produce. However, there may be circumstances in which apatient's wounds have insufficient thrombin. In those cases, theprevious embodiment of the invention should be used.

This embodiment is an advancement over the current technology as itpermits the rapid application of TS to a wound without the time delayassociated with solubilization and mixing of the components. It alsorequires no technical knowledge or skill to operate. Thesecharacteristics make it ideal for use in field applications, such as intrauma packs for soldiers, rescue workers, ambulance/paramedic teams,firemen, in first aid kits for the general public, and by emergency roompersonnel in hospitals. A small version may also be useful for use bythe general public.

EXAMPLE 27 Additional Self-Contained, TS Wound Dressings

The TSs may be formulated as a self-contained wound dressing, or fibrinsealant bandage, which contains the necessary thrombin and fibrinogencomponents of the FG. The self-contained dressing or bandage iseasy-to-use, requiring no advanced technical knowledge or skill tooperate.

The Fibrin Sealant Bandage

The present inventors have prepared a fibrin sealant bandage forapplying a tissue sealing composition to wounded tissue in a patient,wherein the bandage comprises, in order: (1) an occlusive backing; (2) apharmacologically-acceptable adhesive layer on the wound-facing surfaceof the backing; and (3) a layer of dry materials comprising an effectiveamount, in combination, of (a) dry, virally-inactivated, purified tissuefibrinogen complex, (b) dry, virally-inactivated, purified thrombin,affixed to the wound-facing surface of the adhesive layer or backing,and (c) calcium chloride. A removable, waterproof, soft plastic,protective film was placed over the layer of dry materials and theexposed adhesive surface of the bandage for stable storage purposes. Inoperation the waterproof, protective film is removed prior to theapplication of the bandage over the wounded tissue. The bandage wasapplied with pressure until the TS has formed over the target area.

The fibrin sealant bandage was tested using a conventional, adhesivesilicone patch measuring 6 cm×5 cm, having a total area of 30 cm². Thedry components were placed over the adhesive patch to a depth of ½ cm,so that the total volume of fibrin formed by the TS upon hydrationequaled 15 cc (30 cm²×½ cm). The materials used were: 360 mg of topicalfibrinogen complex (TFC), described previously; approximately 340 Uthrombin, also described previously; and 88 mg CaCl₂ (40 mM).

The binding capacity of the bandage for the dry material layer was, inpart, dependent upon applying the dry materials as a uniformly-ground,fine powder. The calcium chloride was ground to a fine powder and mixedwith the finely ground lyophilized TFC and thrombin, and applied as apowder to the adhesive side of the silicone patch and allowed to adhereto form the fibrin sealant patch. In additional versions of the fibrinsealant bandage, the dry materials were mixed and ground together.

Significantly more of the finely ground powder adhered to the siliconepatch when pressure was applied. However, the quantity of dry materialadded to the fibrin sealant bandage was quantifiable. It was found, forexample, in one application using the silicone patch backing that anarea, 2×1 cm², when completely covered by the dry fibrin componentsincreased in weight by 30 mg. This measurement was extrapolated to a dryfibrin component mass per area covered on the backing of 15 mg/cm².

The fibrin sealant patch was applied to a damp cellulose sponge,representative of a tissue wound, so that the fibrin sealant componentwas adjacent to the surface of the sponge. The sponge had beenpreviously dampened with room-temperature distilled H₂O.

Fibrin formation began to develop within 30 seconds of application.Within three minutes of application, a fibrin gel had formed affixingthe tissue sealing fibrin clot to the sponge. This first patch hydratedby the endogenously available liquid was labeled FSB#1.

The previous steps were repeated to prepare patches FSB#2 through FSB#5,however, prior to placing the fibrin sealant bandage against thedampened cellulose sponge, 8 ml of warm PBS were applied to the dryfibrin components affixed to the patch. Incubation of applied patchesFSB#2 through FSB#5 was at 37° C. rather than

TABLE 11 Time Bandage 3′ 5′ 10′ 15′ 20′ 30′ 120′ 180′ 2 clotted clottedclotted clotted clotted clotted clotted clotted 3 in sol'n in sol'n insol'n in sol'n in sol'n in sol'n in sol'n in sol'n 4 in sol'n in sol'nin sol'n in sol'n in sol'n in sol'n in sol'n in sol'n 5 in sol'n veryweak gel weak gel weak, watery gel

TABLE 12 Volume Carbonated Initial Volume Final Volume Experiment # TFCThrombin CaCl₂ H₂O Fibrin Mass Fibrin Mass 1 51.4 mg/ml 57 U/ml 7.1 mM 3mls. 7 mls. 35 mls. 2 30 mg/ml 29 U/ml 7.1 mM 8 mls. 12 mls. 3 60 mg/ml66.7 U/ml 8.3 mM 5 mls. 12 mls. 4 60 mg/ml 58 U/ml 7.1 mM 10 mls. 24mls. 120 mls.room temperature. The results, set forth in Table 11, exemplify the anapplication of the fibrin sealant bandage embodiment wherein the drymaterials are exogenously hydrated prior to application.

Patch FSB#3 was prepared the same as FSB#1, but absent the thrombincomponent. Patch FSB#4 was prepared the same as FSB#1, but absent theTFC component. Patch FSB#5 was prepared the same as FSB#1, but absentthe calcium chloride component. The results of each test were evaluatedover time. As shown below in Table 11, a clotted gel formed when thefibrin components were hydrated with PBS, but remained in solution wheneither the fibrinogen or thrombin components were deleted from fibrinsealant bandage composition. Similarly, although a weak, watery gel wasformed after 30 minutes when the calcium component was deleted from thefibrin sealant bandage and from the hydrating fluid, the composition wasunable to develop into a tissue sealing fibrin clot.

To more clearly visualize the formation of the fibrin clot and theextend to which it bound to adjacent surfaces, a small amount toluidineblue was ground into the powdered fibrin components as a colorindicator.

In practice, with sufficient hydration the silicone patch was easilyremoved from the fibrin clot after hydration of the dry, fibrincomponent layer.

The fibrin sealant bandage, formulated on silicone patches as describedabove, were also found to effectively form fibrin seals when tested ongelatin surfaces and in vivo on rat tissue. Based on the successfulformation of the fibrin seal to a variety of materials and textures,including basic in vivo testing on an uninjured rat, animal studies willbe conducted as described in the previous Examples evaluating the TScomposition to optimize the hemostatic utility of the fibrin sealantbandage, and to establish delivery kinetics of supplementary componentsto be added, e.g., growth hormones, drugs, antibiotics, antiseptics,etc.

The Self-Foaming Fibrin Sealant

The present inventors have prepared a self-foaming fibrin sealantdressing for applying a tissue sealing composition to wounded tissue ina patient, wherein the dressing is applied as an expandable foamcomprising an effective amount, in combination, of (1)virally-inactivated, purified fibrinogen complex, (2)virally-inactivated, purified thrombin, (3) calcium, and (4) aphysiologically acceptable hydration agent; wherein said compositiondoes not significantly inhibit full-thickness skin wound healing. Inpractice, the previously described TS components will be stored in acanister or tank with a pressurized propellant, so that the componentsare delivered to the wound site as an expandable foam, which will withinminute(s) form a fibrin seal.

A bench model test system is prepared from standard Amicon pressurechambers to determine optimal particle size. Particle size has proven tobe important. Preliminary experiments have revealed that a reduction inparticle size of the TFC, fibrin and calcium components results in asignificant reduction in the time required to hydrate the reagents.

Testing is also relevant to determining the feasibility of combining allof the reagents within a single reservoir, or whether it is moreadvantageous to maintain each component in a separate reservoir untilapplication. Although probably more expensive, the latter canisterprototype (having multiple separate reservoirs) may prove advantageous,in terms of stability and long-term storage.

The test system consists of one or two pressure vessels driven by apressurized reservoir containing the pharmaceutically acceptablehydrating agent (e.g., water or PBS), and pressurized compressed gascylinders. The reagents are placed into the appropriate chamber(s) andthe reservoir charged with hydrating agent saturated with the propellantat the desired pressure. Mixing of water and the reagents in theirreservoirs is accomplished by opening connecting valves. The output isdirected into either a single line, or in the case in which thecomponents remain separated, into the joining piece of a Hemedics FibrinSealant Dispenser.

In the present case, the TFC was rehydrated with 3 cc dH₂O, and warmedto 37° C. to the concentrations shown in Table 12. The thrombin wasrehydrated with 0.5 cc CaCl₂ solution (100 mM) to the concentrationsshown in Table 12. The hydrated components were mixed and carbonatedwater (10 cc) was added to produce the volumes shown in Table 12. Theresulting foaming mixture was placed in a vacuum jar to increase thefoaming. Vacuum pressure was applied until the foam dried. The resultwas a permanent, integrated, foamy mass of fibrin, which expandedapproximately 5-fold, and which was both self-adherent and adherent toadjacent textured surfaces.

The foam was also quantitatively measured in calibrated plastic beakers.After two minutes, the volume of the foam was measured and the mass wasgently probed to determine that it had set. The quantitativemeasurements of the expansion of the self-foaming fibrin sealant isindicated in Table 12. Once set, the expandable foam was no longeradhesive to new surfaces.

Based on the successful formation of the self-foaming fibrin dressing,animal studies will be conducted as described in the previous Examplesevaluating the TS composition to optimize the hemostatic utility of theself-foam fibrin sealant dressing, and to establish delivery kinetics ofsupplementary components to be added, e.g., growth hormones, drugs,antibiotics, etc.

Other embodiments of the invention will be apparent to those of skill inthe art from a consideration of this specification or practice of theinvention disclosed herein. Since modifications will be apparent tothose of skill in the art, it is intended that this invention be limitedonly by the scope of the appended claims.

1. A method of using a supplemented tissue sealant composition toprevent or treat a disease in a patient by releasing into said patientan amount of a supplement effective for prevention or treatment of saiddisease, said method comprising: (a) preparing a supplemented tissuesealant composition comprising: (i) an effective amount of demineralizedbone matrix as a supplement, and (ii) fibrinogen, or a derivative ormetabolite thereof, in an amount which is capable of forming a fibrinmatrix; and (b) applying said supplemented tissue sealant composition ofstep (a) to a patient in need thereof at a concentration which releasesinto said patient an effective amount of said supplement to prevent ortreat said disease; wherein said fibrinogen forms a fibrin matrix whenin the presence of thrombin and Ca⁺⁺ and water, further wherein saidsupplemented tissue sealant composition adheres to the tissue of saidpatient, further wherein said supplement is released from said fibrinmatrix into the external environment of use for a sustained period,further wherein the amount of said supplement in said composition isgreater than the amount of said supplement which is soluble in saidfibrin matrix, and further wherein said sustained period is greater thanthe period obtained according to simple diffusion kinetics, upondissolution of the fibrin matrix.
 2. A method of using a supplementedtissue sealant composition to reverse or halt progression of adisease-state in a patient suffering from said disease by releasing intosaid patient an amount of said supplement effective for reversing orhalting progression of said disease-state, said method comprising: (a)preparing a supplemented tissue sealant composition comprising: (i) aneffective amount of demineralized bone matrix as a supplement, and (ii)fibrinogen, or a derivative or metabolite thereof, in an amount which iscapable of forming a fibrin matrix; and (b) applying said supplementedtissue sealant composition of step (a) to a patient in need thereof at aconcentration which releases into said patient an effective amount ofsaid supplement to reverse or halt progression of said disease-state;wherein said fibrinogen forms a fibrin matrix when in the presence ofthrombin and Ca⁺⁺ and water, further wherein said supplemented tissuesealant composition adheres to the tissue of said patient, furtherwherein said supplement is released from said fibrin matrix into theexternal environment of use for a sustained period, further wherein theamount of said supplement in said composition is greater than the amountof said supplement which is soluble in said fibrin matrix, and furtherwherein said sustained period is greater than the period obtainedaccording to simple diffusion kinetics, upon dissolution of the fibrinmatrix.
 3. A method of using a supplemented tissue sealant compositionto prevent activation of a disease-state in a patient predisposed tosaid disease by releasing into said patient an amount of said supplementeffective for preventing activation of said disease-state, said methodcomprising: (a) preparing a supplemented tissue sealant compositioncomprising: (i) an effective amount of demineralized bone matrix as asupplement, and (ii) fibrinogen, or a derivative or metabolite thereof,in an amount which is capable of forming a fibrin matrix; and (b)applying said supplemented tissue sealant composition of step (a) to apatient in need thereof at a concentration which releases into saidpatient an effective amount of said supplement to prevent activation ofsaid disease-state; wherein said fibrinogen forms a fibrin matrix whenin the presence of thrombin and Ca⁺⁺ and water, further wherein saidsupplemented tissue sealant composition adheres to the tissue of saidpatient, further wherein said supplement is released from said fibrinmatrix into the external environment of use for a sustained period,further wherein the amount of said supplement in said composition isgreater than the amount of said supplement which is soluble in saidfibrin matrix, and further wherein said sustained period is greater thanthe period obtained according to simple diffusion kinetics, upondissolution of the fibrin matrix.
 4. A method of using a supplementedtissue sealant composition to prevent or treat a disease in a patient byreleasing into said patient an amount of a supplement effective forprevention or treatment of said disease, said method comprising: (a)preparing a supplemented tissue sealant composition comprising: (i) aneffective amount of a bone morphogenetic protein as a supplement, and(ii) fibrinogen, or a derivative or metabolite thereof, in an amountwhich is capable of forming a fibrin matrix; and (b) applying saidsupplemented tissue sealant composition of step (a) to a patient in needthereof at a concentration which releases into said patient an effectiveamount of said supplement to prevent or treat said disease; wherein saidfibrinogen forms a fibrin matrix when in the presence of thrombin andCa⁺⁺ and water, further wherein said supplemented tissue sealantcomposition adheres to the tissue of said patient, further wherein saidsupplement is released from said fibrin matrix into the externalenvironment of use for a sustained period, and further wherein saidsustained period is greater than the period obtained according to simplediffusion kinetics, upon dissolution of the fibrin matrix.
 5. A methodof using a supplemented tissue sealant composition to reverse or haltprogression of a disease-state in a patient suffering from said diseaseby releasing into said patient an amount of said supplement effectivefor reversing or halting progression of said disease-state, said methodcomprising: (a) preparing a supplemented tissue sealant compositioncomprising: (i) an effective amount of a bone morphogenetic protein as asupplement, and (ii) fibrinogen, or a derivative or metabolite thereof,in an amount which is capable of forming a fibrin matrix; and (b)applying said supplemented tissue sealant composition of step (a) to apatient in need thereof at a concentration which releases into saidpatient an effective amount of said supplement to reverse or haltprogression of said disease-state; wherein said fibrinogen forms afibrin matrix when in the presence of thrombin and Ca⁺⁺ and water,further wherein said supplemented tissue sealant composition adheres tothe tissue of said patient, further wherein said supplement is releasedfrom said fibrin matrix into the external environment of use for asustained period, further wherein the amount of said supplement in saidcomposition is greater than the amount of said supplement which issoluble in said fibrin matrix, and further wherein said sustained periodis greater than the period obtained according to simple diffusionkinetics, upon dissolution of the fibrin matrix.
 6. A method of using asupplemented tissue sealant composition to prevent activation of adisease-state in a patient predisposed to said disease by releasing intosaid patient an amount of said supplement effective for preventingactivation of said disease-state, said method comprising: (a) preparinga supplemented tissue sealant composition comprising: (i) an effectiveamount of a bone morphogenetic protein as a supplement, and (ii)fibrinogen, or a derivative or metabolite thereof, in an amount which iscapable of forming a fibrin matrix; and (b) applying said supplementedtissue sealant composition of step (a) to a patient in need thereof at aconcentration which releases into said patient an effective amount ofsaid supplement to prevent activation of said disease-state; whereinsaid fibrinogen forms a fibrin matrix when in the presence of thrombinand Ca⁺⁺ and water, further wherein said supplemented tissue sealantcomposition adheres to the tissue of said patient, further wherein saidsupplement is released from said fibrin matrix into the externalenvironment of use for a sustained period, and further wherein saidsustained period is greater than the period obtained according to simplediffusion kinetics, upon dissolution of the fibrin matrix.
 7. The methodof any one of claims 1, 2–3, 4, 5 or 6, wherein said supplemented tissuesealant composition is applied to tissue that is injured or wounded. 8.The method of any one of claims 1, 2–3, 4, 5 or 6, wherein saidsupplemented tissue sealant composition is applied to bone and/orcartilage.
 9. The method of claim 8, wherein said composition furthercomprises an effective amount of at least one antimicrobial composition.10. The method of claim 8, wherein said disease is osteomyelitis. 11.The method of any one of claims 1, 2–3, 4, 5 or 6, wherein saidsupplemented tissue sealant composition is applied to teeth and/ortissue of the mouth or gums.
 12. The method of claim 11, wherein saiddisease is periodontitis.
 13. The method of any one of claims 1, 2–3, 4,5 or 6, wherein said supplemented tissue sealant composition furthercomprises thrombin.
 14. The method of any one of claims 1, 2–3, 4, 5 or6, wherein said supplemented tissue sealant composition furthercomprises Factor XIII.
 15. The method of any one of claims 1, 2–3, 4, 5or 6, wherein said fibrinogen is recombinantly-produced humanfibrinogen.
 16. The method of claim 13, wherein said thrombin isrecombinantly-produced human thrombin.
 17. The method of claim 14,wherein said Factor XIII is recombinantly-produced human Factor XIII.18. The method of any one of claims 1, 2–3, 4, 5 or 6, wherein saidsupplemented tissue sealant composition further comprises Ca⁺⁺.
 19. Themethod of claim 1, wherein said supplement interacts with said fibrinmatrix to permit sustained-release of said supplement.
 20. The method ofclaim 1, wherein said supplement is introduced into said tissue sealantcomposition in a solution, suspension or emulsion said solution,suspension or emulsion having a higher rate of dissolution or diffusionin said fibrin matrix than said supplement contained therein, so thatsaid supplement is deposited within said fibrin matrix as a solidprecipitate.
 21. The method of claim 1, wherein said supplement isintroduced into said tissue sealant composition in solution in a carrierwhich is of sufficiently low solubility to permit sustained release ofsaid supplement.
 22. The method of claim 21, wherein said supplement isintroduced into said tissue sealant composition as an emulsion.
 23. Themethod of claim 1, wherein said supplement is introduced into saidtissue sealant composition in a carrier, said carrier interacting withsaid fibrin matrix and said supplement to permit sustained release ofsaid supplement.
 24. The method of claim 2, wherein said supplementinteracts with said fibrin matrix to permit sustained-release of saidsupplement.
 25. The method of claim 2, wherein said supplement isintroduced into said tissue sealant composition in a solution,suspension or emulsion, said solution, suspension or emulsion having ahigher rate of dissolution or diffusion in said fibrin matrix than saidsupplement contained therein, so that said supplement is depositedwithin said fibrin matrix as a solid precipitate.
 26. The method ofclaim 2, wherein said supplement is introduced into said tissue sealantcomposition in solution in a carrier which is of sufficiently lowsolubility to permit sustained release of said supplement.
 27. Themethod of claim 26, wherein said supplement is introduced into saidtissue sealant composition as an emulsion.
 28. The method of claim 2,wherein said supplement is introduced into said tissue sealantcomposition in a carrier, said carrier interacting with said fibrinmatrix and said supplement to permit sustained release of saidsupplement.
 29. The method of claim 3, wherein said supplement interactswith said fibrin matrix to permit sustained-release of said supplement.30. The method of claim 3, wherein said supplement is introduced intosaid tissue sealant composition in a solution, suspension or emulsion,said solution, suspension or emulsion having a higher rate ofdissolution or diffusion in said fibrin matrix than said supplementcontained therein, so that said supplement is deposited within saidfibrin matrix as a solid precipitate.
 31. The method of claim 3, whereinsaid supplement is introduced into said tissue sealant composition insolution in a carrier which is of sufficiently low solubility to permitsustained release of said supplement.
 32. The method of claim 31,wherein said supplement is introduced into said tissue sealantcomposition as an emulsion.
 33. The method of claim 3, wherein saidsupplement is introduced into said tissue sealant composition in acarrier, said carrier interacting with said fibrin matrix and saidsupplement to permit sustained release of said supplement.